Antibody therapeutics that bind LAG3

ABSTRACT

There is disclosed compositions and methods relating to or derived from anti-LAG3 antibodies. More specifically, there is disclosed fully human antibodies that bind LAG3, LAG3-antibody binding fragments and derivatives of such antibodies, and LAG3-binding polypeptides comprising such fragments. Further still, there is disclosed nucleic acids encoding such antibodies, antibody fragments and derivatives and polypeptides, cells comprising such polynucleotides, methods of making such antibodies, antibody fragments and derivatives and polypeptides, and methods of using such antibodies, antibody fragments and derivatives and polypeptides, including methods of treating a disease.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/195,651 filed on Jul. 22, 2015, the entire contents of which areincorporated by reference in their entirety herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 20, 2016, isnamed 126036-04702_SL.txt and is 21,129 bytes in size.

TECHNICAL FIELD

The present disclosure provides compositions and methods relating to orderived from anti-LAG3 antibodies. More specifically, the presentdisclosure provides fully human antibodies that bind LAG3, LAG3-antibodybinding fragments and derivatives of such antibodies, and LAG3-bindingpolypeptides comprising such fragments. Further still, the presentdisclosure provides antibody fragments and derivatives and polypeptides,cells comprising such polynucleotides, methods of making suchantibodies, antibody fragments and derivatives and polypeptides, andmethods of using such antibodies, antibody fragments and derivatives andpolypeptides, including methods of treating a disease.

BACKGROUND

Lymphocyte Activation Gene-3, or LAG3 (also known as CD223), is a memberof the immunoglobulin supergene family and is structurally andgenetically related to CD4. LAG3 is not expressed on resting peripheralblood lymphocytes but is expressed on activated T cells and NK cells.LAG3 is a membrane protein encoded by a gene located on the distal partof the short arm of chromosome 12, near the CD4 gene, suggesting thatthe LAG3 gene may have evolved through gene duplication (Triebel et al.(1990) J. Exp. Med. 171:1393-1405).

Similar to CD4, LAG3 has been demonstrated to interact with MHC Class IImolecules but, unlike CD4, LAG3 does not interact with the humanimmunodeficiency virus gp120 protein (Baixeras et al. (1992) J. Exp.Med. 176:327-337). Studies using a soluble LAG3 immunoglobulin fusionprotein (sLAG3Ig) demonstrated direct and specific binding of LAG3 toMHC class II on the cell surface (Huard et al. (1996) Eur. J. Immunol.26:1180-1186).

In in vitro studies of antigen-specific T cell responses, the additionof anti-LAG3 antibodies led to increased T cell proliferation, higherexpression of activation antigens such as CD25, and higherconcentrations of cytokines such as interferon-gamma and interleukin-4,supporting a role for the LAG3/MHC class II interaction indown-regulating antigen-dependent stimulation of CD4⁺ T lymphocytes(Huard et al. (1994) Eur. J. Immunol. 24:3216-3221). Theintra-cytoplasmic region of LAG3 has been demonstrated to interact witha protein termed LAP, which is thought to be a signal transductionmolecule involved in the downregulation of the CD3/TCR activationpathway (Iouzalen et al. (2001) Eur. J. Immunol. 31:2885-2891).Furthermore, CD4⁺CD25⁺ regulatory T cells (T_(reg)) have been shown toexpress LAG3 upon activation and antibodies to LAG3 inhibit suppressionby induced T_(reg) cells, both in vitro and in vivo, suggesting thatLAG3 contributes to the suppressor activity of T_(reg) cells (Huang, C.et al. (2004) Immunity 21:503-513). Still further, LAG3 has been shownto negatively regulate T cell homeostasis by regulatory T cells in bothT cell-dependent and independent mechanisms (Workman and Vignali (2005)J. Immunol. 174:688-695).

In certain circumstances, LAG3 also has been shown to haveimmunostimulatory effects. For example, LAG3 transfected tumor cellstransplanted into syngeneic mice showed growth reduction or completeregression as compared to untransfected tumor cells, suggesting thatLAG3 expression on the tumor cells stimulated an anti-tumor response bytriggering antigen LAG3 presenting cells via MHC class II molecules(Prigent et al. (1999) Eur. J. Immunol. 29:3867-3876). Additionally,soluble LAG3 Ig fusion protein has been shown to stimulate both humoraland cellular immune responses when administered to mice together with anantigen, indicating that soluble LAG3Ig can function as a vaccineadjuvant (El Mir and Triebel (2000) J. Immunol. 164:5583-5589).Furthermore, soluble human LAG3Ig has been shown to amplify in vitrogeneration of type I tumor-specific immunity (Casati et al. (2006)Cancer Res. 66:4450-4460). The functional activity of LAG3 is reviewedfurther in Triebel (2003) Trends Immunol. 24:619-622. In view of theabove, additional agents for modulating the activity of LAG3 are ofinterest.

SUMMARY OF THE INVENTION

The present invention provides novel anti-human LAG3 (hLAG3) antibodiesand fragments thereof. The present invention relates to anti-LAG3antibodies that are advantageous, for example, in that they can act asimmune checkpoint inhibitors and may be used in immunotherapy fortreating disorders such as cancer.

In one embodiment, the present disclosure provides a fully humanantibody of an IgG class that binds to a LAG3 epitope with a bindingaffinity of at least 10⁻⁶ M, which has a heavy chain variable domainsequence that is at least 95% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 8, SEQ IDNO. 10, SEQ ID NO. 12, and combinations thereof, and has a light chainvariable domain sequence that is at least 95% identical to an amino acidsequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO.3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9,SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, and combinations thereof.In one embodiment, the fully human antibody has both a heavy chain and alight chain wherein the antibody has a heavy chain/light chain variabledomain sequence selected from the group consisting SEQ ID NO. 1/SEQ IDNO. 2 (called L35D4 herein), SEQ ID NO. 1/SEQ ID NO. 3 (called L35G6herein), SEQ ID NO. 1/SEQ ID NO. 4 (called L33H11 herein), SEQ ID NO.1/SEQ ID NO. 5 (called L32A9 herein), SEQ ID NO. 1/SEQ ID NO. 6 (calledL32D10 herein), SEQ ID NO. 1/SEQ ID NO. 7 (called L32A4 herein), SEQ IDNO. 8/SEQ ID NO. 9 (called L3A1 herein), SEQ ID NO. 10/SEQ ID NO. 11(called L3A10 herein), SEQ ID NO. 12/SEQ ID NO. 13 (called L3C5 herein),SEQ ID NO. 8/SEQ ID NO. 14 (called L3E3 herein), and combinationsthereof.

In one embodiment, the present disclosure provides a Fab fully humanantibody fragment, having a variable domain region from a heavy chainand a variable domain region from a light chain, wherein the heavy chainvariable domain sequence is at least 95% identical to an amino acidsequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.8, SEQ ID NO. 10, SEQ ID NO. 12, and combinations thereof, and has alight chain variable domain sequence that is at least 95% identical toan amino acid sequence selected from the group consisting of SEQ ID NO.2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7,SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, andcombinations thereof. In one embodiment, the fully human antibody Fabfragment has both a heavy chain variable domain region and a light chainvariable domain region wherein the antibody has a heavy chain/lightchain variable domain sequence selected from the group consisting of SEQID NO. 1/SEQ ID NO. 2, SEQ ID NO. 1/SEQ ID NO. 3, SEQ ID NO. 1/SEQ IDNO. 4, SEQ ID NO. 1/SEQ ID NO. 5, SEQ ID NO. 1/SEQ ID NO. 6, SEQ ID NO.1/SEQ ID NO. 7, SEQ ID NO. 8/SEQ ID NO. 9, SEQ ID NO. 10/SEQ ID NO. 11,SEQ ID NO. 12/SEQ ID NO. 13, and SEQ ID NO. 8/SEQ ID NO. 14, andcombinations thereof.

In one embodiment, the present disclosure provides a single chain humanantibody, having a variable domain region from a heavy chain and avariable domain region from a light chain and a peptide linkerconnecting the heavy chain and light chain variable domain regions,wherein the heavy chain variable domain sequence is at least 95%identical to an amino acid sequence selected from the group consistingof SEQ ID NO. 1, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, and has thelight chain variable domain sequence is at least 95% identical to anamino acid sequence selected from the group consisting of SEQ ID NO. 2,SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7,SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, andcombinations thereof. In one embodiment, the fully human single chainantibody has both a heavy chain variable domain region and a light chainvariable domain region, wherein the single chain fully human antibodyhas a heavy chain/light chain variable domain sequence selected from thegroup consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 1/SEQ ID NO.3, SEQ ID NO. 1/SEQ ID NO. 4, SEQ ID NO. 1/SEQ ID NO. 5, SEQ ID NO.1/SEQ ID NO. 6, SEQ ID NO. 1/SEQ ID NO. 7, SEQ ID NO. 8/SEQ ID NO. 9,SEQ ID NO. 10/SEQ ID NO. 11, SEQ ID NO. 12/SEQ ID NO. 13, and SEQ ID NO.8/SEQ ID NO. 14, and combinations thereof.

In one embodiment, the present disclosure further provides a method fortreating a broad spectrum of mammalian cancers, infectious diseases orautoimmune reactions, comprising administering an anti-LAG3 polypeptide,wherein the fully human antibody has a heavy chain variable domainsequence that is at least 95% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 8, SEQ IDNO. 10, SEQ ID NO. 12, and combinations thereof, and has a light chainvariable domain sequence that is at least 95% identical to an amino acidsequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO.3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9,SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, and combinations thereof.

In one embodiment, the Fab fully human antibody fragment has the heavychain variable domain sequence that is at least 95% identical to anamino acid sequence selected from the group consisting of SEQ ID NO. 1,SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, and combinations thereof,and has the light chain variable domain sequence that is at least 95%identical to an amino acid sequence selected from the group consistingof SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6,SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14,and combinations thereof.

In one embodiment, the single chain human antibody has a heavy chainvariable domain sequence that is at least 95% identical to an amino acidsequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.8, SEQ ID NO. 10, SEQ ID NO. 12, and combinations thereof, and has alight chain variable domain sequence that is at least 95% identical toan amino acid sequence selected from the group consisting of SEQ ID NO.2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7,SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, andcombinations thereof.

In one embodiment, the fully human antibody has both a heavy chainvariable domain region and a light chain variable domain region, whereinthe antibody has a heavy chain/light chain variable domain sequenceselected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2 (calledL35D4 herein), SEQ ID NO. 1/SEQ ID NO. 3 (called L35G6 herein), SEQ IDNO. 1/SEQ ID NO. 4 (called L33H11 herein), SEQ ID NO. 1/SEQ ID NO. 5(called L32A9 herein), SEQ ID NO. 1/SEQ ID NO. 6 (called L32D10 herein),SEQ ID NO. 1/SEQ ID NO. 7 (called L32A4 herein), SEQ ID NO. 8/SEQ ID NO.9 (called L3A1 herein), SEQ ID NO. 10/SEQ ID NO. 11 (called L3A10herein), SEQ ID NO. 12/SEQ ID NO. 13 (called L3C5 herein), SEQ ID NO.8/SEQ ID NO. 14 (called L3E3 herein), and combinations thereof. In oneembodiment, the fully human single chain antibody has both a heavy chainvariable domain region and a light chain variable domain region, whereinthe single chain fully human antibody has a heavy chain/light chainvariable domain sequence selected from the group consisting of SEQ IDNO. 1/SEQ ID NO. 2, SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 1/SEQ ID NO.3, SEQ ID NO. 1/SEQ ID NO. 4, SEQ ID NO. 1/SEQ ID NO. 5, SEQ ID NO.1/SEQ ID NO. 6, SEQ ID NO. 1/SEQ ID NO. 7, SEQ ID NO. 8/SEQ ID NO. 9,SEQ ID NO. 10/SEQ ID NO. 11, SEQ ID NO. 12/SEQ ID NO. 13, SEQ ID NO.8/SEQ ID NO. 14, and combinations thereof.

In one embodiment, the broad spectrum of mammalian cancers, infectiousdiseases, or autoimmune reactions to be treated is selected from thegroup consisting of non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma(BL), multiple myeloma (MM), B chronic lymphocytic leukemia (B-CLL), Band T acute lymphocytic leukemia (ALL), T cell lymphoma (TCL), acutemyeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's Lymphoma(HL), chronic myeloid leukemia (CML) non-Hodgkin's lymphoma (NHL), acutelymphocytic leukemia (ALL), acute myeloid leukemia (AML), chroniclymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiplemyeloma (MM), breast cancer, ovarian cancer, head and neck cancer,bladder cancer, melanoma, colorectal cancer, pancreatic cancer, lungcancer, leiomyoma, leiomyosarcoma, glioma, glioblastoma, and solidtumors, wherein solid tumors are selected from the group consisting ofbreast tumors, ovarian tumors, lung tumors, pancreatic tumors, prostatetumors, melanoma tumors, colorectal tumors, lung tumors, head and necktumors, bladder tumors, esophageal tumors, liver tumors, and kidneytumors.

In one embodiment, the invention provides an isolated fully humanantibody of an IgG class that binds to a LAG3 epitope, said antibodycomprising: a heavy chain variable domain sequence that is at least 95%identical to an amino acid sequence selected from the group consistingof SEQ ID NO. 1, SEQ ID NO. 8, SEQ ID NO. 10, and SEQ ID NO. 12; and alight chain variable domain sequence that is at least 95% identical toan amino acid sequence selected from the group consisting of SEQ ID NO.2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7,SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14.

In one embodiment, the fully human antibody comprises a heavychain/light chain variable domain sequence selected from the groupconsisting of SEQ ID NO. 1/SEQ ID NO. 2 (L35D4), SEQ ID NO. 1/SEQ ID NO.3 (L35G6), SEQ ID NO. 1/SEQ ID NO. 4 (L33H11), SEQ ID NO. 1/SEQ ID NO. 5(L32A9), SEQ ID NO. 1/SEQ ID NO. 6 (L32D10), SEQ ID NO. 1/SEQ ID NO. 7(L32A4), SEQ ID NO. 8/SEQ ID NO. 9 (L3A1), SEQ ID NO. 10/SEQ ID NO. 11(L3A10), SEQ ID NO. 12/SEQ ID NO. 13 (L3C5), and SEQ ID NO. 8/SEQ ID NO.14 (L3E3).

In one embodiment, the invention features an anti-LAG3 fully humanantibody Fab fragment, comprising a heavy chain variable domain sequencethat is at least 95% identical to an amino acid sequence selected fromthe group consisting of SEQ ID NO. 1, SEQ ID NO. 8, SEQ ID NO. 10, andSEQ ID NO. 12; and comprising a light chain variable domain sequencethat is at least 95% identical to an amino acid sequence selected fromthe group consisting of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ IDNO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ IDNO. 13, and SEQ ID NO. 14. In one embodiment, the fully human antibodyFab fragment comprises a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO.2, SEQ ID NO. 1/SEQ ID NO. 3, SEQ ID NO. 1/SEQ ID NO. 4, SEQ ID NO.1/SEQ ID NO. 5, SEQ ID NO. 1/SEQ ID NO. 6, SEQ ID NO. 1/SEQ ID NO. 7,SEQ ID NO. 8/SEQ ID NO. 9, SEQ ID NO. 10/SEQ ID NO. 11, SEQ ID NO.12/SEQ ID NO. 13, and SEQ ID NO. 8/SEQ ID NO. 14.

In one embodiment, the present invention provides an anti-LAG3 singlechain human antibody comprising a heavy chain variable domain and alight chain variable domain which are connected by a peptide linker,wherein the heavy chain variable domain comprises an amino acid sequencethat is at least 95% identical to an amino acid sequence selected fromthe group consisting of SEQ ID NO. 1, SEQ ID NO. 8, SEQ ID NO. 10, andSEQ ID NO. 12; and the light chain variable domain comprises an aminoacid sequence that is at least 95% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 3, SEQ IDNO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9, SEQ IDNO. 11, SEQ ID NO. 13, and SEQ ID NO. 14.

In one embodiment, the single chain fully human antibody comprises aheavy chain/light chain variable domain sequence selected from the groupconsisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 1/SEQ ID NO. 3, SEQID NO. 1/SEQ ID NO. 4, SEQ ID NO. 1/SEQ ID NO. 5, SEQ ID NO. 1/SEQ IDNO. 6, SEQ ID NO. 1/SEQ ID NO. 7, SEQ ID NO. 8/SEQ ID NO. 9, SEQ ID NO.10/SEQ ID NO. 11, SEQ ID NO. 12/SEQ ID NO. 13, and SEQ ID NO. 8/SEQ IDNO. 14.

In one embodiment, the invention provides an isolated anti-human LAG3(hLAG3) antibody, or an antigen-binding fragment thereof, comprising aheavy chain variable domain comprising complementarity determiningregions (CDRs) as set forth in the heavy chain variable domain aminoacid sequence selected from the group consisting of SEQ ID NO. 1, SEQ IDNO. 8, SEQ ID NO. 10, and SEQ ID NO. 12; and comprising a light chainvariable domain comprising CDRs as set forth in a light chain variableregion amino acid sequence selected from the group consisting of SEQ IDNO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ IDNO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, and SEQ ID NO. 14. Inone embodiment, the heavy chain variable domain comprises an amino acidsequence that is at least 95% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 8, SEQ IDNO. 10, and SEQ ID NO. 12; and comprises a light chain variable domaincomprising an amino acid sequence that is at least 95% identical to anamino acid sequence selected from the group consisting of SEQ ID NO. 2,SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7,SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14. In oneembodiment, the heavy chain variable domain comprises an amino acidsequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.8, SEQ ID NO. 10, and SEQ ID NO. 12; and comprises a light chainvariable domain comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO.5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.13, SEQ ID NO. 14.

In another embodiment, the present invention features an isolatedanti-human LAG3 (hLAG3) antibody, or an antigen-binding fragmentthereof, comprising a heavy chain variable domain comprising a heavychain CDR set (CDR1, CDR2, and CDR3) selected from the group consistingof SEQ ID Nos: 15, 16, and 17; SEQ ID Nos: 36, 37, and 38; SEQ ID Nos:42, 43, and 44; and SEQ ID Nos: 48, 49, and 50; and a light chainvariable domain comprising a light chain CDR set (CDR1, CDR2, and CDR3)selected from the group consisting of SEQ ID Nos: 18, 19, and 20; SEQ IDNos: 21, 22, and 23; SEQ ID Nos: 24, 25, and 26; SEQ ID Nos: 27, 28, and29; SEQ ID Nos: 30, 31, and 32; SEQ ID Nos: 33, 34, and 35; SEQ ID Nos:39, 40, and 41; SEQ ID Nos: 45, 46, and 47; SEQ ID Nos: 51, 52, and 53;and SEQ ID Nos: 54, 55, and 56.

In one embodiment, the antibody comprises a heavy chain CDR set/lightchain CDR set selected from the group consisting of the heavy chainvariable domain CDR set of SEQ ID Nos: 15, 16, and 17, and the lightchain variable domain CDR set of 18, 19, and 20; the heavy chainvariable domain CDR set of SEQ ID Nos: 15, 16, and 17, and the lightchain variable domain CDR set of 21, 22, and 23; the heavy chainvariable domain CDR set of SEQ ID Nos: 15, 16, and 17, and the lightchain variable domain CDR set of 24, 25, and 26; the heavy chainvariable domain CDR set of SEQ ID Nos: 15, 16, and 17, and the lightchain variable domain CDR set of 27, 28, and 29; the heavy chainvariable domain CDR set of SEQ ID Nos: 15, 16, and 17, and the lightchain variable domain CDR set of 30, 31, and 32; the heavy chainvariable domain CDR set of SEQ ID Nos: 15, 16, and 17, and the lightchain variable domain CDR set of 33, 34, and 35; the heavy chainvariable domain CDR set of SEQ ID Nos: 36, 37, and 38, and the lightchain variable domain CDR set of 39, 40, and 41; the heavy chainvariable domain CDR set of SEQ ID Nos: 42, 43, and 44, and the lightchain variable domain CDR set of 45, 46, and 47; the heavy chainvariable domain CDR set of SEQ ID Nos: 48, 49, and 50, and the lightchain variable domain CDR set of 51, 52, and 53; and the heavy chainvariable domain CDR set of SEQ ID Nos: 36, 37, and 38, and the lightchain variable domain CDR set of 54, 55, and 56.

In one embodiment, an anti-LAG3 antibody or antibody fragment may beused in a method for treating a subject having cancer, an infectiousdisease, or an autoimmune disease, said method comprising administeringan effective amount of the anti-LAG3 antibody or antibody fragment tothe subject.

In one embodiment, the cancer is selected from the group consisting ofnon-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), multiple myeloma(MM), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphocyticleukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML),hairy cell leukemia (HCL), Hodgkin's Lymphoma (HL), chronic myeloidleukemia (CML), melanoma, renal cancer, prostate cancer, breast cancer,colon cancer, and lung cancer.

In another embodiment, the cancer is selected from the group consistingof bone cancer, pancreatic cancer, skin cancer, cancer of the head orneck, cutaneous or intraocular malignant melanoma, uterine cancer,ovarian cancer, rectal cancer, cancer of the anal region, stomachcancer, testicular cancer, carcinoma of the fallopian tubes, carcinomaof the endometrium, carcinoma of the cervix, carcinoma of the vagina,carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma,cancer of the esophagus, cancer of the small intestine, cancer of theendocrine system, cancer of the thyroid gland, cancer of the parathyroidgland, cancer of the adrenal gland, sarcoma of soft tissue, cancer ofthe urethra, cancer of the penis, chronic or acute leukemias includingacute myeloid leukemia, chronic myeloid leukemia, acute lymphoblasticleukemia, chronic lymphocytic leukemia, solid tumors of childhood,lymphocytic lymphoma, cancer of the bladder, cancer of the kidney orureter, carcinoma of the renal pelvis, neoplasm of the central nervoussystem (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axistumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma,environmentally induced cancer, and cancer induced by asbestos.

In another embodiment, the cancer is metastatic cancer that expressesPD-L1.

In one embodiment, the infectious disease is selected from the groupconsisting of HIV, Hepatitis (A, B, & C), Influenza, Herpes, Giardia,Malaria, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa,flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus,respiratory syncytial virus, mumps virus, rotavirus, measles virus,rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus,papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, andarboviral encephalitis virus.

In another embodiment, the infectious disease is selected from the groupconsisting of chlamydia, rickettsial bacteria, mycobacteria,staphylococci, streptococci, pneumonococci, meningococci and gonococci,klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria,salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague,leptospirosis, and Lyme disease bacteria.

In another embodiment, the infectious disease is selected from the groupconsisting of Entamoeba histolytica, Balantidium coli, Naegleriafowleri,Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystiscarinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei,Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, andNippostrongylus brasiliensis.

In one embodiment, the autoimmune disease is selected from the groupconsisting of Alzheimer's disease, allergy, asthma, celiac disease,Crohn's disease, Grave's disease, inflammatory bowel disease (IBD),lupus, multiple sclerosis, Myasthenia Gravis, polymyalgia rheumatica,rheumatoid arthritis, type I diabetes, and vasculitis.

In certain embodiments, the anti-LAG3 antibody, or antigen-bindingfragment thereof, of the invention has a binding affinity (K_(D)) of atleast 1×10⁻⁶ M. In other embodiments, the antibody, or antigen-bindingfragment thereof, of the invention has a K_(D) of at least 1×10⁻⁷ M. Inother embodiments, the antibody, or antigen-binding fragment thereof, ofthe invention has a K_(D) of at least 1×10⁻⁸ M.

In certain embodiments, the antibody is an IgG1 isotype. In otherembodiments, the antibody is an IgG4 isotype.

In one embodiment, the antibody, or antigen-binding fragment, describedherein is recombinant. In another embodiment, the antibody, orantigen-binding fragment, described herein, is a recombinant humanantibody, or antigen binding fragment of an antibody.

In one embodiment, the invention provides a pharmaceutical compositioncomprising an effective amount of an anti-LAG3 antibody, or antibodyfragment disclosed herein, and a pharmaceutically acceptable carrier.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph that shows that several anti-LAG3 antibodies hadreactivity with activated T cells, shown as % positive cells.

FIG. 2 is a graph that shows the cross-reactivity of anti-hLAG3antibodies L35G6, L33H11, L35D4, L32A9, L32A4, and L32D10 to recombinantmouse LAG3 and human LAG3. Anti-AIP antibody C7 was used as a control.

FIG. 3 shows results that determined the effect of anti-LAG3 antibodieson LAG3 expressing T cells. A percent change with respect to the mediumcontrol was calculated and is shown in FIG. 3. An isotype match IgG wasused as a control (cIg).

FIG. 4 provides a graph that shows the results of in vitro studies usingmixed lymphocyte reactions (MLR) to measure T cell activation. Cellswere assayed for CD25 expression as a measure of T cell activation (%CD25 positive).

FIG. 5 is a graph that shows the results of an ELISA assay to determinethe effect of anti-LAG3 antibodies L32D10, L3E3, L3C5 and L3A1 (atconcentrations of 5 μg/ml and 0.5 μg/ml) on IL-2 cytokine production.

FIG. 6 is a graph that shows the results of an ELISA assay to determinethe effect of anti-LAG3 antibodies L32D10, L3E3, L3C5 and L3A1 (atconcentrations of 5 μg/ml and 0.5 μg/ml) on interferon gamma (IFNγ)cytokine production.

DETAILED DESCRIPTION Definitions

The terms “peptide,” “polypeptide” and “protein” each refers to amolecule comprising two or more amino acid residues joined to each otherby peptide bonds. These terms encompass, e.g., native and artificialproteins, protein fragments and polypeptide analogs (such as muteins,variants, and fusion proteins) of a protein sequence as well aspost-translationally, or otherwise covalently or non-covalently,modified proteins. A peptide, polypeptide, or protein may be monomericor polymeric.

A “variant” of a polypeptide (for example, an antibody) comprises anamino acid sequence wherein one or more amino acid residues are insertedinto, deleted from and/or substituted into the amino acid sequencerelative to another polypeptide sequence. Disclosed variants include,for example, fusion proteins.

A “derivative” of a polypeptide is a polypeptide (e.g., an antibody)that has been chemically modified, e.g., via conjugation to anotherchemical moiety (such as, for example, polyethylene glycol or albumin,e.g., human serum albumin), phosphorylation, and glycosylation.

Unless otherwise indicated, the term “antibody” includes, in addition toantibodies comprising two full-length heavy chains (each chaincomprising a variable region and a constant region) and two full-lengthlight chains (each chain comprising a variable region and a constantregion), derivatives, variants, fragments, and muteins thereof, examplesof which are described below.

An “antigen binding protein” is a protein comprising a portion thatbinds to an antigen and, optionally, a scaffold or framework portionthat allows the antigen binding portion to adopt a conformation thatpromotes binding of the antigen binding protein to the antigen. Examplesof antigen binding proteins include antibodies, antibody fragments(e.g., an antigen binding portion of an antibody), antibody derivatives,and antibody analogs. The antigen binding protein can comprise, forexample, an alternative protein scaffold or artificial scaffold withgrafted CDRs or CDR derivatives. Such scaffolds include, but are notlimited to, antibody-derived scaffolds comprising mutations introducedto, for example, stabilize the three-dimensional structure of theantigen binding protein as well as wholly synthetic scaffoldscomprising, for example, a biocompatible polymer. See, for example,Korndorfer et al., 2003, Proteins: Structure, Function, andBioinformatics, Volume 53, Issue 1:121-129; Roque et al., 2004,Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics(“PAMs”) can be used, as well as scaffolds based on antibody mimeticsutilizing fibronection components as a scaffold.

An antigen binding protein can have, for example, the structure of anaturally occurring immunoglobulin, such as an IgG. An “immunoglobulinG” (or IgG) is a tetrameric molecule. In a naturally occurring IgG, eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion (or domain) of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The carboxy-terminal portion ofeach chain defines a constant region primarily responsible for effectorfunction. Human light chains are classified as kappa or lambda lightchains. Heavy chains are classified as mu, delta, gamma, alpha, orepsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, andIgE, respectively. Preferably, the anti-LAG3 antibodies disclosed hereinare characterized by their variable domain sequences in the heavy VH andlight VL amino acid sequences. Within light and heavy chains, thevariable and constant regions are joined by a “J” region of about 12 ormore amino acids, with the heavy chain also including a “D” region ofabout 10 more amino acids. See generally, Fundamental Immunology Ch. 7(Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). The variable regionsof each light/heavy chain pair form the antibody binding site such thatan intact immunoglobulin has two binding sites.

The variable regions of naturally occurring immunoglobulin chainsexhibit the same general structure of relatively conserved frameworkregions (FR) joined by three hypervariable regions, also calledcomplementarity determining regions or CDRs. From N-terminus toC-terminus, both light and heavy chains comprise the domains FR1, CDR1,FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to eachdomain can be in accordance with the definitions of Kabat et al. inSequences of Proteins of Immunological Interest, 5th Ed., US Dept. ofHealth and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991.Other numbering systems for the amino acids in immunoglobulin chainsinclude IMGT® (international ImMunoGeneTics information system; Lefrancet al, Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger andPluckthun, J. Mol. Biol. 309(3):657-670; 2001).

In one embodiment, an “antibody” refers to an intact immunoglobulin,such as an IgG, or to an antigen binding portion thereof that competeswith the intact antibody for specific binding, unless otherwisespecified. In one embodiment, an intact antibody is an IgG1, IgG2, IgG3or IgG4. Heavy and light chain variable domain sequences and CDRs may beselected from those described herein in SEQ ID Nos: 1 to 14 and SEQ IDNos: 15 to 56, respectively.

The term “monospecific”, as used herein, refers to an antibody thatdisplays an affinity for one particular epitope. Monospecific antibodypreparations can be made up of about 10%, 20%, 30%, 40%, 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 97%, 99%, or 99.9% antibody having specificbinding activity for the particular antigen.

An “antibody fragment”, “antigen binding portion of an antibody” or“antigen binding fragment of an antibody” comprises a portion of anintact antibody, and preferably comprises the antibody antigen bindingor variable domains. Examples of an antibody fragment include a Fab, anFab′, an F(ab′)2, an Fv fragment, and a linear antibody.

A Fab fragment is a monovalent fragment having the V_(L), V_(H), C_(L)and C_(H1) domains; a F(ab′)₂ fragment is a bivalent fragment having twoFab fragments linked by a disulfide bridge at the hinge region; a Fdfragment has the V_(H) and C_(H1) domains; an Fv fragment has the V_(L)and V_(H) domains of a single arm of an antibody; and a dAb fragment hasa V_(H) domain, a V_(L), domain, or an antigen-binding fragment of aV_(H) or V_(L) domain (U.S. Pat. Nos. 6,846,634; 6,696,245, US App.Pub.20/0202512; 2004/0202995; 2004/0038291; 2004/0009507; 2003/0039958,and Ward et al., Nature 341:544-546, 1989).

A single-chain antibody (scFv) is an antibody in which a V_(L) and aV_(H) region are joined via a linker (e.g., a synthetic sequence ofamino acid residues) to form a continuous protein chain wherein thelinker is long enough to allow the protein chain to fold back on itselfand form a monovalent antigen binding site (see, e.g., Bird et al.,1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci.USA 85:5879-83).

Diabodies are bivalent antibodies comprising two polypeptide chains,wherein each polypeptide chain comprises VH and VL domains joined by alinker that is too short to allow for pairing between two domains on thesame chain, thus allowing each domain to pair with a complementarydomain on another polypeptide chain (see, e.g., Holliger et al., 1993,Proc. Natl. Acad. Sci. USA 90:6444-48, and Poljak et al., 1994,Structure 2:1121-23). If the two polypeptide chains of a diabody areidentical, then a diabody resulting from their pairing will have twoidentical antigen binding sites. Polypeptide chains having differentsequences can be used to make a diabody with two different antigenbinding sites. Similarly, tribodies and tetrabodies are antibodiescomprising three and four polypeptide chains, respectively, and formingthree and four antigen binding sites, respectively, which can be thesame or different.

An antigen binding protein, such as an antibody, may have one or morebinding sites. If there is more than one binding site, the binding sitesmay be identical to one another or may be different. For example, anaturally occurring human immunoglobulin typically has two identicalbinding sites, while a “bispecific” or “bifunctional” antibody has twodifferent binding sites.

The term “human antibody” includes all antibodies that have one or morevariable and constant regions derived from human immunoglobulinsequences. In one embodiment, all of the variable and constant domainsof the antibody are derived from human immunoglobulin sequences(referred to as “a fully human antibody”). These antibodies may beprepared in a variety of ways, examples of which are described below,including through the immunization with an antigen of interest of amouse that is genetically modified to express antibodies derived fromhuman heavy and/or light chain-encoding genes. In a preferredembodiment, a fully human antibody is made using recombinant methods.

A “humanized antibody” has a sequence that differs from the sequence ofan antibody derived from a non-human species by one or more amino acidsubstitutions, deletions, and/or additions, such that the humanizedantibody is less likely to induce an immune response, and/or induces aless severe immune response, as compared to the non-human speciesantibody, when it is administered to a human subject. In one embodiment,certain amino acids in the framework and constant domains of the heavyand/or light chains of the non-human species antibody are mutated toproduce the humanized antibody. In another embodiment, the constantdomain(s) from a human antibody are fused to the variable domain(s) of anon-human species. In another embodiment, one or more amino acidresidues in one or more CDR sequences of a non-human antibody arechanged to reduce the likely immunogenicity of the non-human antibodywhen it is administered to a human subject, wherein the changed aminoacid residues either are not critical for immunospecific binding of theantibody to its antigen, or the changes to the amino acid sequence thatare made are conservative changes, such that the binding of thehumanized antibody to the antigen is not significantly worse than thebinding of the non-human antibody to the antigen. Examples of how tomake humanized antibodies may be found in U.S. Pat. Nos. 6,054,297,5,886,152 and 5,877,293.

The term “chimeric antibody” refers to an antibody that contains one ormore regions from one antibody and one or more regions from one or moreother antibodies. In one embodiment, one or more of the CDRs are derivedfrom a human anti-LAG3 antibody. In another embodiment, all of the CDRsare derived from a human anti-LAG3 antibody. In another embodiment, theCDRs from more than one human anti-LAG3 antibodies are mixed and matchedin a chimeric antibody. For instance, a chimeric antibody may comprise aCDR1 from the light chain of a first human anti-PAR-2 antibody, a CDR2and a CDR3 from the light chain of a second human anti-LAG3 antibody,and the CDRs from the heavy chain from a third anti-LAG3 antibody. Othercombinations are possible.

Further, the framework regions may be derived from one of the sameanti-LAG3 antibodies, from one or more different antibodies, such as ahuman antibody, or from a humanized antibody. In one example of achimeric antibody, a portion of the heavy and/or light chain isidentical with, homologous to, or derived from an antibody from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is/are identical with,homologous to, or derived from an antibody (-ies) from another speciesor belonging to another antibody class or subclass. Also included arefragments of such antibodies that exhibit the desired biologicalactivity (i.e., the ability to specifically bind LAG3).

A “CDR grafted antibody” is an antibody comprising one or more CDRsderived from an antibody of a particular species or isotype and theframework of another antibody of the same or different species orisotype.

A “multi-specific antibody” is an antibody that recognizes more than oneepitope on one or more antigens. A subclass of this type of antibody isa “bi-specific antibody” which recognizes two distinct epitopes on thesame or different antigens.

An antigen binding protein “specifically binds” to an antigen (e.g.,human LAG3) if it binds to the antigen with a dissociation constant of 1nanomolar or less.

An “antigen binding domain,” “antigen binding region,” or “antigenbinding site” is a portion of an antigen binding protein that containsamino acid residues (or other moieties) that interact with an antigenand contribute to the antigen binding proteins specificity and affinityfor the antigen. For an antibody that specifically binds to its antigen,this will include at least part of at least one of its CDR domains.

The term “Fc polypeptide” includes native and mutein forms ofpolypeptides derived from the Fc region of an antibody. Truncated formsof such polypeptides containing the hinge region that promotesdimerization also are included. Fusion proteins comprising Fc moieties(and oligomers formed therefrom) offer the advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns.

An “epitope” is the portion of a molecule that is bound by an antigenbinding protein (e.g., by an antibody). An epitope can comprisenon-contiguous portions of the molecule (e.g., in a polypeptide, aminoacid residues that are not contiguous in the polypeptide's primarysequence but that, in the context of the polypeptide's tertiary andquaternary structure, are near enough to each other to be bound by anantigen binding protein).

The “percent identity” or “percent homology” of two polynucleotide ortwo polypeptide sequences is determined by comparing the sequences usingthe GAP computer program (a part of the GCG Wisconsin Package, version10.3 (Accelrys, San Diego, Calif.)) using its default parameters.

The terms “polynucleotide,” “oligonucleotide” and “nucleic acid” areused interchangeably throughout and include DNA molecules (e.g., cDNA orgenomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNAgenerated using nucleotide analogs (e.g., peptide nucleic acids andnon-naturally occurring nucleotide analogs), and hybrids thereof. Thenucleic acid molecule can be single-stranded or double-stranded. In oneembodiment, the nucleic acid molecules of the invention comprise acontiguous open reading frame encoding an antibody, or a fragment,derivative, mutein, or variant thereof.

Two single-stranded polynucleotides are “the complement” of each otherif their sequences can be aligned in an anti-parallel orientation suchthat every nucleotide in one polynucleotide is opposite itscomplementary nucleotide in the other polynucleotide, without theintroduction of gaps, and without unpaired nucleotides at the 5′ or the3′ end of either sequence. A polynucleotide is “complementary” toanother polynucleotide if the two polynucleotides can hybridize to oneanother under moderately stringent conditions. Thus, a polynucleotidecan be complementary to another polynucleotide without being itscomplement.

A “vector” is a nucleic acid that can be used to introduce anothernucleic acid linked to it into a cell. One type of vector is a“plasmid,” which refers to a linear or circular double stranded DNAmolecule into which additional nucleic acid segments can be ligated.Another type of vector is a viral vector (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), whereinadditional DNA segments can be introduced into the viral genome. Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors comprising a bacterialorigin of replication and episomal mammalian vectors). Other vectors(e.g., non-episomal mammalian vectors) are integrated into the genome ofa host cell upon introduction into the host cell, and thereby arereplicated along with the host genome. An “expression vector” is a typeof vector that can direct the expression of a chosen polynucleotide.

A nucleotide sequence is “operably linked” to a regulatory sequence ifthe regulatory sequence affects the expression (e.g., the level, timing,or location of expression) of the nucleotide sequence. A “regulatorysequence” is a nucleic acid that affects the expression (e.g., thelevel, timing, or location of expression) of a nucleic acid to which itis operably linked. The regulatory sequence can, for example, exert itseffects directly on the regulated nucleic acid, or through the action ofone or more other molecules (e.g., polypeptides that bind to theregulatory sequence and/or the nucleic acid). Examples of regulatorysequences include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Further examples of regulatorysequences are described in, for example, Goeddel, 1990, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.and Baron et al., 1995, Nucleic Acids Res. 23:3605-06.

A “host cell” is a cell that can be used to express a nucleic acid,e.g., a nucleic acid of the invention. A host cell can be a prokaryote,for example, E. coli, or it can be a eukaryote, for example, asingle-celled eukaryote (e.g., a yeast or other fungus), a plant cell(e.g., a tobacco or tomato plant cell), an animal cell (e.g., a humancell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or aninsect cell) or a hybridoma. Examples of host cells include the COS-7line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al., 1981,Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinesehamster ovary (CHO) cells or their derivatives such as Veggie CHO andrelated cell lines which grow in serum-free media (see Rasmussen et al.,1998, Cytotechnology 28:31) or CHO strain DX-B11, which is deficient inDHFR (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20),HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1/EBNA cell line derivedfrom the African green monkey kidney cell line CV1 (ATCC CCL 70) (seeMcMahan et al., 1991, EMBO J. 10:2821), human embryonic kidney cellssuch as 293,293 EBNA or MSR 293, human epidermal A431 cells, humanColo205 cells, other transformed primate cell lines, normal diploidcells, cell strains derived from in vitro culture of primary tissue,primary explants, HL-60, U937, HaK or Jurkat cells. In one embodiment, ahost cell is a mammalian host cell, but is not a human host cell.Typically, a host cell is a cultured cell that can be transformed ortransfected with a polypeptide-encoding nucleic acid, which can then beexpressed in the host cell. The phrase “recombinant host cell” can beused to denote a host cell that has been transformed or transfected witha nucleic acid to be expressed. A host cell also can be a cell thatcomprises the nucleic acid but does not express it at a desired levelunless a regulatory sequence is introduced into the host cell such thatit becomes operably linked with the nucleic acid. It is understood thatthe term host cell refers not only to the particular subject cell butalso to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to, e.g., mutationor environmental influence, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

The term “recombinant antibody” refers to an antibody that is expressedfrom a cell or cell line transfected with one or more expression vectorscomprising the coding sequence of the antibody, where said codingsequence is not naturally associated with the cell. In one embodiment, arecombinant antibody has a glycosylation pattern that is different thanthe glycosylation pattern of an antibody having the same sequence if itwere to exist in nature. In one embodiment, a recombinant antibody isexpressed in a mammalian host cell which is not a human host cell.Notably, individual mammalian host cells have unique glycosylationpatterns.

The term “effective amount” as used herein, refers to that amount of anantibody, or an antigen binding portion thereof that binds LAG3, whichis sufficient to effect treatment, prognosis or diagnosis of a diseaseassociated with LAG3 dependent signaling, as described herein, whenadministered to a subject. Therapeutically effective amounts ofantibodies provided herein, when used alone or in combination, will varydepending upon the relative activity of the antibodies and combinations(e.g., in inhibiting cell growth) and depending upon the subject anddisease condition being treated, the weight and age of the subject, theseverity of the disease condition, the manner of administration and thelike, which can readily be determined by one of ordinary skill in theart.

The term “isolated” refers to a protein (e.g., an antibody) that issubstantially free of other cellular material and/or chemicals. In oneembodiment, an isolated antibody is expressed by a cell from a differentspecies, e.g., a human antibody expressed in a CHO cell, and issubstantially free of other proteins from the different species. Aprotein may be rendered substantially free of naturally associatedcomponents (or components associated with the cellular expression systemused to produce the antibody) by isolation, using protein purificationtechniques well known in the art. In one embodiment, the antibodies, orantigen binding fragments, of the invention are isolated.

A “neutralizing antibody” or an “inhibitory antibody” is an antibodythat inhibits the proteolytic activation of LAG3 when an excess of theanti-LAG3 antibody reduces the amount of activation by at least about20% using an assay such as those described herein in the Examples. Invarious embodiments, the antigen binding protein reduces the amount ofamount of proteolytic activation of LAG3 by at least 30%, 40%, 50%, 60%,70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, and 99.9%.

LAG3 Antigen Binding Proteins

The present invention pertains to LAG3 binding proteins, particularlyanti-LAG3 antibodies, or antigen-binding portions thereof, and usesthereof. Various aspects of the invention relate to antibodies andantibody fragments, pharmaceutical compositions, nucleic acids,recombinant expression vectors, and host cells for making suchantibodies and fragments. Methods of using the antibodies of theinvention to detect human LAG3, to inhibit LAG3 activity, either invitro or in vivo, and to prevent or treat disorders such as cancer arealso encompassed by the invention.

As described in Table 3 below, included in the invention are novel humanantibody heavy and light chain variable regions and CDRs that arespecific to human LAG3.

In one embodiment, the invention provides an anti-LAG3 antibody, or anantigen-binding fragment thereof, that comprises a heavy chain having avariable domain comprising an amino acid sequence as set forth in anyone of SEQ ID NO. 1, SEQ ID NO. 8, SEQ ID NO. 10, and SEQ ID NO. 12. Inone embodiment, the invention provides an anti-LAG3 antibody, or anantigen-binding fragment thereof, that comprises a light chain having avariable domain comprising an amino acid sequence as set forth in anyone of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ IDNO. 6, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13 and SEQID NO. 14. In one embodiment, the invention provides an anti-LAG3antibody, or an antigen-binding fragment thereof, that comprises a lightchain having a variable domain comprising an amino acid sequence as setforth in any one of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO.5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.13 and SEQ ID NO. 14; and a heavy chain having a variable domaincomprising an amino acid sequence as set forth in any one of SEQ ID NO.1, SEQ ID NO. 8, SEQ ID NO. 10, and SEQ ID NO. 12.

In one embodiment, the present disclosure provides a fully humanantibody of an IgG class that binds to a LAG3 epitope with a bindingaffinity of at least 10⁻⁶ M, which has a heavy chain variable domainsequence which is at least 95% identical, at least 96% identical, atleast 97% identical, at least 98% identical, or at least 99% identicalto the amino acid sequences selected from the group consisting of SEQ IDNO. 1, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, and combinationsthereof, and that has a light chain variable domain sequence that is atleast 95% identical, at least 96% identical, at least 97% identical, atleast 98% identical, or at least 99% identical to the amino acidsequence consisting of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ IDNO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ IDNO. 13, SEQ ID NO. 14, and combinations thereof.

In one embodiment, the fully human antibody has both a heavy chain and alight chain wherein the antibody has a heavy chain/light chain variabledomain sequence selected from the group consisting of SEQ ID NO. 1/SEQID NO. 2 (called L35D4 herein), SEQ ID NO. 1/SEQ ID NO. 3 (called L35G6herein), SEQ ID NO. 1/SEQ ID NO. 4 (called L33H11 herein), SEQ ID NO.1/SEQ ID NO. 5 (called L32A9 herein), SEQ ID NO. 1/SEQ ID NO. 6 (calledL32D10 herein), SEQ ID NO. 1/SEQ ID NO. 7 (called L32A4 herein), SEQ IDNO. 8/SEQ ID NO. 9 (called L3A1 herein), SEQ ID NO. 10/SEQ ID NO. 11(called L3A10 herein), SEQ ID NO. 12/SEQ ID NO. 13 (called L3C5 herein),SEQ ID NO. 8/SEQ ID NO. 14 (called L3E3 herein), and combinationsthereof.

Complementarity determining regions (CDRs) are known as hypervariableregions both in the light chain and the heavy chain variable domains ofan antibody. The more highly conserved portions of variable domains arecalled the framework (FR). Complementarity determining regions (CDRs)and framework regions (FR) of a given antibody may be identified usingsystems known in the art, such as those described by Kabat et al. supra;Lefranc et al., supra and/or Honegger and Pluckthun, supra. For example,the numbering system described in Kabat et al. (1991, NIH Publication91-3242, National Technical Information Service, Springfield, Va.) iswell known to those in the art. Kabat et al. defined a numbering systemfor variable domain sequences that is applicable to any antibody. One ofordinary skill in the art can unambiguously assign this system of “Kabatnumbering” to any variable domain amino acid sequence, without relianceon any experimental data beyond the sequence itself.

In certain embodiments, the present invention provides an anti-LAG3antibody comprising the CDRs of the heavy and light chain variabledomains described in Table 3 (SEQ ID Nos: 1 to 14). For example, theinvention provides an anti-LAG3 antibody, or antigen-binding fragmentthereof, comprising a heavy chain variable region having the CDRsdescribed in an amino acid sequence as set forth in any one of SEQ IDNO. 1, SEQ ID NO. 8, SEQ ID NO. 10 and SEQ ID NO. 12. In one embodiment,the invention provides an anti-LAG3 antibody, or antigen-bindingfragment thereof, comprising a light chain variable region having theCDRs described in an amino acid sequence as set forth in any one of SEQID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ IDNO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13 and SEQ ID NO. 14. Inone embodiment, the invention provides an anti-LAG3 antibody, orantigen-binding fragment thereof, comprising a light chain variableregion having the CDRs described in an amino acid sequence as set forthin any one of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5,SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13and SEQ ID NO. 14; and a heavy chain variable region having the CDRsdescribed in an amino acid sequence as set forth in any one of SEQ IDNO. 1, SEQ ID NO. 8, SEQ ID NO. 10 and SEQ ID NO. 12.

In one embodiment, the present invention features an isolated anti-humanLAG3 (hLAG3) antibody, or an antigen-binding fragment thereof,comprising a heavy chain variable domain comprising a heavy chain CDRset (CDR1, CDR2, and CDR3) selected from the group consisting of SEQ IDNos: 15, 16, and 17; SEQ ID Nos: 36, 37, and 38; SEQ ID Nos: 42, 43, and44; and SEQ ID Nos: 48, 49, and 50; and a light chain variable domaincomprising a light chain CDR set (CDR1, CDR2, and CDR3) selected fromthe group consisting of SEQ ID Nos: 18, 19, and 20; SEQ ID Nos: 21, 22,and 23; SEQ ID Nos: 24, 25, and 26; SEQ ID Nos: 27, 28, and 29; SEQ IDNos: 30, 31, and 32; SEQ ID Nos: 33, 34, and 35; SEQ ID Nos: 39, 40, and41; SEQ ID Nos: 45, 46, and 47; SEQ ID Nos: 51, 52, and 53; and SEQ IDNos: 54, 55, and 56.

In one embodiment, the antibody of the invention comprises a heavy chainCDR set/light chain CDR set selected from the group consisting of theheavy chain variable domain CDR set of SEQ ID Nos: 15, 16, and 17, andthe light chain variable domain CDR set of 18, 19, and 20; the heavychain variable domain CDR set of SEQ ID Nos: 15, 16, and 17, and thelight chain variable domain CDR set of 21, 22, and 23; the heavy chainvariable domain CDR set of SEQ ID Nos: 15, 16, and 17, and the lightchain variable domain CDR set of 24, 25, and 26; the heavy chainvariable domain CDR set of SEQ ID Nos: 15, 16, and 17, and the lightchain variable domain CDR set of 27, 28, and 29; the heavy chainvariable domain CDR set of SEQ ID Nos: 15, 16, and 17, and the lightchain variable domain CDR set of 30, 31, and 32; the heavy chainvariable domain CDR set of SEQ ID Nos: 15, 16, and 17, and the lightchain variable domain CDR set of 33, 34, and 35; the heavy chainvariable domain CDR set of SEQ ID Nos: 36, 37, and 38, and the lightchain variable domain CDR set of 39, 40, and 41; the heavy chainvariable domain CDR set of SEQ ID Nos: 42, 43, and 44, and the lightchain variable domain CDR set of 45, 46, and 47; the heavy chainvariable domain CDR set of SEQ ID Nos: 48, 49, and 50, and the lightchain variable domain CDR set of 51, 52, and 53; and the heavy chainvariable domain CDR set of SEQ ID Nos: 36, 37, and 38, and the lightchain variable domain CDR set of 54, 55, and 56.

In one embodiment, the invention provides an anti-LAG3 antibody, or anantigen-binding fragment thereof, comprising a heavy chain comprising aCDR3 domain as set forth in any one of SEQ ID NO. 1, SEQ ID NO. 8, SEQID NO. 10 or SEQ ID NO. 12, and comprising a variable domain comprisingan amino acid sequence that has at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to a sequence as set forthin any one of SEQ ID NO. 1, SEQ ID NO. 8, SEQ ID NO. 10 or SEQ ID NO.12. In one embodiment, the invention provides an anti-LAG3 antibody, oran antigen-binding fragment thereof, comprising a light chain comprisinga CDR3 domain as set forth in any one of SEQ ID NO.2, SEQ ID NO.3, SEQID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO. 7, SEQ ID NO. 9, SEQ IDNO.11, SEQ ID NO.13 or SEQ ID NO.14, and having a light chain variabledomain comprising an amino acid sequence that has at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical to a sequenceas set forth in any one of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ IDNO.5, SEQ ID NO.6, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO.11, SEQ IDNO.13 and SEQ ID NO.14. Thus, in certain embodiments, the CDR3 domain isheld constant, while variability may be introduced into the remainingCDRs and/or framework regions of the heavy and/or light chains, whilethe antibody, or antigen binding fragment thereof, retains the abilityto bind to LAG3 and retains the functional characteristics, e.g.,binding affinity, of the parent.

One or more CDRs may be incorporated into a molecule either covalentlyor noncovalently to make it an antigen binding protein.

An antigen binding protein may incorporate the CDR(s) as part of alarger polypeptide chain, may covalently link the CDR(s) to anotherpolypeptide chain, or may incorporate the CDR(s) noncovalently. The CDRspermit the antigen binding protein to specifically bind to a particularantigen of interest.

In one embodiment, the substitutions made within a heavy or light chainthat is at least 95% identical (or at least 96% identical, or at least97% identical, or at least 98% identical, or at least 99% identical) areconservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which an amino acid residue is substituted byanother amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of similarity may be adjustedupwards to correct for the conservative nature of the substitution.Means for making this adjustment are well-known to those of skill in theart. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, hereinincorporated by reference. Examples of groups of amino acids that haveside chains with similar chemical properties include (1) aliphatic sidechains: glycine, alanine, valine, leucine and isoleucine; (2)aliphatic-hydroxyl side chains: serine and threonine; (3)amide-containing side chains: asparagine and glutamine; (4) aromaticside chains: phenylalanine, tyrosine, and tryptophan; (5) basic sidechains: lysine, arginine, and histidine; (6) acidic side chains:aspartate and glutamate, and (7) sulfur-containing side chains arecysteine and methionine.

In one embodiment, the present invention is directed to an antibody, oran antigen binding fragment thereof, having the antigen binding regionsof any of the antibodies described in Table 3.

In one embodiment, the present invention is directed to an antibody, oran antigen binding fragment thereof, having antigen binding regions ofantibody L35D4. In one embodiment, the invention provides an antibody,or antigen-binding fragment thereof, comprising a heavy chain variabledomain sequence as set forth in SEQ ID NO: 1, and a light chain variabledomain sequence as set forth in SEQ ID NO: 2. In one embodiment, theinvention is directed to an antibody having a heavy chain variabledomain comprising the CDRs of SEQ ID NO: 1, and a light chain variabledomain comprising the CDRs of SEQ ID NO: 2. In one embodiment, theinvention features an isolated human antibody, or antigen-bindingfragment thereof, that comprises a heavy chain variable region having anamino acid sequence that is at least 95% identical, at least 96%identical, at least 97% identical, at least 98% identical, or at least99% identical to the sequence set forth in SEQ ID NO: 1, and comprises alight chain variable region having an amino acid sequence that is atleast 95% identical, at least 96% identical, at least 97% identical, atleast 98% identical, or at least 99% identical to the sequence set forthin SEQ ID NO: 2. In one embodiment, the invention features an anti-LAG3antibody, or an antigen-binding portion thereof, comprising a heavychain variable region comprising a CDR3 domain comprising the amino acidas set forth in SEQ ID NO: 17, a CDR2 domain comprising the amino acidsequence as set forth in SEQ ID NO: 16, and a CDR1 domain comprising theamino acid sequence as set forth in SEQ ID NO: 15; and comprising alight chain variable region comprising a CDR3 domain comprising theamino acid as set forth in SEQ ID NO: 20, a CDR2 domain comprising theamino acid sequence as set forth in SEQ ID NO: 19, and a CDR1 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 18. Theantibody may further be an IgG1 or an IgG4 isotype.

In one embodiment, the present invention is directed to an antibody, oran antigen binding fragment thereof, having antigen binding regions ofantibody L35G6. In one embodiment, the invention provides an antibody,or antigen-binding fragment thereof, comprising a heavy chain variabledomain sequence as set forth in SEQ ID NO: 1, and a light chain variabledomain sequence as set forth in SEQ ID NO: 3. In one embodiment, theinvention is directed to an antibody having a heavy chain variabledomain comprising the CDRs of SEQ ID NO: 1, and a light chain variabledomain comprising the CDRs of SEQ ID NO:3. In one embodiment, theinvention features an isolated human antibody, or antigen-bindingfragment thereof, that comprises a heavy chain variable region having anamino acid sequence that is at least 95% identical, at least 96%identical, at least 97% identical, at least 98% identical, or at least99% identical to the sequence set forth in SEQ ID NO: 1, and comprises alight chain variable region having an amino acid sequence that is atleast 95% identical, at least 96% identical, at least 97% identical, atleast 98% identical, or at least 99% identical to the sequence set forthin SEQ ID NO: 3. In one embodiment, the invention features an anti-LAG3antibody, or an antigen-binding portion thereof, comprising a heavychain variable region comprising a CDR3 domain comprising the amino acidas set forth in SEQ ID NO: 17, a CDR2 domain comprising the amino acidsequence as set forth in SEQ ID NO: 16, and a CDR1 domain comprising theamino acid sequence as set forth in SEQ ID NO: 15; and comprising alight chain variable region comprising a CDR3 domain comprising theamino acid as set forth in SEQ ID NO: 23, a CDR2 domain comprising theamino acid sequence as set forth in SEQ ID NO: 22, and a CDR1 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 21. Theantibody may further be an IgG1 or an IgG4 isotype.

In one embodiment, the present invention is directed to an antibody, oran antigen binding fragment thereof, having antigen binding regions ofantibody L33H11. In one embodiment, the invention provides an antibody,or antigen-binding fragment thereof, comprising a heavy chain variabledomain sequence as set forth in SEQ ID NO: 1, and a light chain variabledomain sequence as set forth in SEQ ID NO: 4. In one embodiment, theinvention is directed to an antibody having a heavy chain variabledomain comprising the CDRs of SEQ ID NO: 1, and a light chain variabledomain comprising the CDRs of SEQ ID NO: 4. In one embodiment, theinvention features an isolated human antibody, or antigen-bindingfragment thereof, that comprises a heavy chain variable region having anamino acid sequence that is at least 95% identical, at least 96%identical, at least 97% identical, at least 98% identical, or at least99% identical to the sequence set forth in SEQ ID NO: 1, and comprises alight chain variable region having an amino acid sequence that is atleast 95% identical, at least 96% identical, at least 97% identical, atleast 98% identical, or at least 99% identical to the sequence set forthin SEQ ID NO: 4. In one embodiment, the invention features an anti-LAG3antibody, or an antigen-binding portion thereof, comprising a heavychain variable region comprising a CDR3 domain comprising the amino acidas set forth in SEQ ID NO: 17, a CDR2 domain comprising the amino acidsequence as set forth in SEQ ID NO: 16, and a CDR1 domain comprising theamino acid sequence as set forth in SEQ ID NO: 15; and comprising alight chain variable region comprising a CDR3 domain comprising theamino acid as set forth in SEQ ID NO: 26, a CDR2 domain comprising theamino acid sequence as set forth in SEQ ID NO: 25, and a CDR1 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 24. Theantibody may further be an IgG1 or an IgG4 isotype.

In one embodiment, the present invention is directed to an antibody, oran antigen binding fragment thereof, having antigen binding regions ofantibody L32A9. In one embodiment, the invention provides an antibody,or antigen-binding fragment thereof, comprising a heavy chain variabledomain sequence as set forth in SEQ ID NO: 1, and a light chain variabledomain sequence as set forth in SEQ ID NO: 5. In one embodiment, theinvention is directed to an antibody having a heavy chain variabledomain comprising the CDRs of SEQ ID NO: 1, and a light chain variabledomain comprising the CDRs of SEQ ID NO: 5. In one embodiment, theinvention features an isolated human antibody, or antigen-bindingfragment thereof, that comprises a heavy chain variable region having anamino acid sequence that is at least 95% identical, at least 96%identical, at least 97% identical, at least 98% identical, or at least99% identical to the sequence set forth in SEQ ID NO: 1, and comprises alight chain variable region having an amino acid sequence that is atleast 95% identical, at least 96% identical, at least 97% identical, atleast 98% identical, or at least 99% identical to the sequence set forthin SEQ ID NO: 5. In one embodiment, the invention features an anti-LAG3antibody, or an antigen-binding portion thereof, comprising a heavychain variable region comprising a CDR3 domain comprising the amino acidas set forth in SEQ ID NO: 17, a CDR2 domain comprising the amino acidsequence as set forth in SEQ ID NO: 16, and a CDR1 domain comprising theamino acid sequence as set forth in SEQ ID NO: 15; and comprising alight chain variable region comprising a CDR3 domain comprising theamino acid as set forth in SEQ ID NO: 29, a CDR2 domain comprising theamino acid sequence as set forth in SEQ ID NO: 28, and a CDR1 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 27. Theantibody may further be an IgG1 or an IgG4 isotype.

In one embodiment, the present invention is directed to an antibody, oran antigen binding fragment thereof, having antigen binding regions ofantibody L32D10. In one embodiment, the invention provides an antibody,or antigen-binding fragment thereof, comprising a heavy chain variabledomain sequence as set forth in SEQ ID NO: 1, and a light chain variabledomain sequence as set forth in SEQ ID NO: 6. In one embodiment, theinvention is directed to an antibody having a heavy chain variabledomain comprising the CDRs of SEQ ID NO: 1, and a light chain variabledomain comprising the CDRs of SEQ ID NO: 6. In one embodiment, theinvention features an isolated human antibody, or antigen-bindingfragment thereof, that comprises a heavy chain variable region having anamino acid sequence that is at least 95% identical, at least 96%identical, at least 97% identical, at least 98% identical, or at least99% identical to the sequence set forth in SEQ ID NO: 1, and comprises alight chain variable region having an amino acid sequence that is atleast 95% identical, at least 96% identical, at least 97% identical, atleast 98% identical, or at least 99% identical to the sequence set forthin SEQ ID NO: 6. In one embodiment, the invention features an anti-LAG3antibody, or an antigen-binding portion thereof, comprising a heavychain variable region comprising a CDR3 domain comprising the amino acidas set forth in SEQ ID NO: 17, a CDR2 domain comprising the amino acidsequence as set forth in SEQ ID NO: 16, and a CDR1 domain comprising theamino acid sequence as set forth in SEQ ID NO: 15; and comprising alight chain variable region comprising a CDR3 domain comprising theamino acid as set forth in SEQ ID NO: 32, a CDR2 domain comprising theamino acid sequence as set forth in SEQ ID NO: 31, and a CDR1 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 30. Theantibody may further be an IgG1 or an IgG4 isotype.

In one embodiment, the present invention is directed to an antibody, oran antigen binding fragment thereof, having antigen binding regions ofantibody L32A4. In one embodiment, the invention provides an antibody,or antigen-binding fragment thereof, comprising a heavy chain variabledomain sequence as set forth in SEQ ID NO: 1, and a light chain variabledomain sequence as set forth in SEQ ID NO: 7. In one embodiment, theinvention is directed to an antibody having a heavy chain variabledomain comprising the CDRs of SEQ ID NO: 1, and a light chain variabledomain comprising the CDRs of SEQ ID NO: 7. In one embodiment, theinvention features an isolated human antibody, or antigen-bindingfragment thereof, that comprises a heavy chain variable region having anamino acid sequence that is at least 95% identical, at least 96%identical, at least 97% identical, at least 98% identical, or at least99% identical to the sequence set forth in SEQ ID NO: 1, and comprises alight chain variable region having an amino acid sequence that is atleast 95% identical, at least 96% identical, at least 97% identical, atleast 98% identical, or at least 99% identical to the sequence set forthin SEQ ID NO: 7. In one embodiment, the invention features an anti-LAG3antibody, or an antigen-binding portion thereof, comprising a heavychain variable region comprising a CDR3 domain comprising the amino acidas set forth in SEQ ID NO: 17, a CDR2 domain comprising the amino acidsequence as set forth in SEQ ID NO: 16, and a CDR1 domain comprising theamino acid sequence as set forth in SEQ ID NO: 15; and comprising alight chain variable region comprising a CDR3 domain comprising theamino acid as set forth in SEQ ID NO: 35, a CDR2 domain comprising theamino acid sequence as set forth in SEQ ID NO: 34, and a CDR1 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 33. Theantibody may further be an IgG1 or an IgG4 isotype.

In one embodiment, the present invention is directed to an antibody, oran antigen binding fragment thereof, having antigen binding regions ofantibody L3A1. In one embodiment, the invention provides an antibody, orantigen-binding fragment thereof, comprising a heavy chain variabledomain sequence as set forth in SEQ ID NO: 8, and a light chain variabledomain sequence as set forth in SEQ ID NO: 9. In one embodiment, theinvention is directed to an antibody having a heavy chain variabledomain comprising the CDRs of SEQ ID NO: 8, and a light chain variabledomain comprising the CDRs of SEQ ID NO: 9. In one embodiment, theinvention features an isolated human antibody, or antigen-bindingfragment thereof, that comprises a heavy chain variable region having anamino acid sequence that is at least 95% identical, at least 96%identical, at least 97% identical, at least 98% identical, or at least99% identical to the sequence set forth in SEQ ID NO: 8, and comprises alight chain variable region having an amino acid sequence that is atleast 95% identical, at least 96% identical, at least 97% identical, atleast 98% identical, or at least 99% identical to the sequence set forthin SEQ ID NO: 9. In one embodiment, the invention features an anti-LAG3antibody, or an antigen-binding portion thereof, comprising a heavychain variable region comprising a CDR3 domain comprising the amino acidas set forth in SEQ ID NO: 38, a CDR2 domain comprising the amino acidsequence as set forth in SEQ ID NO: 37, and a CDR1 domain comprising theamino acid sequence as set forth in SEQ ID NO: 36; and comprising alight chain variable region comprising a CDR3 domain comprising theamino acid as set forth in SEQ ID NO: 41, a CDR2 domain comprising theamino acid sequence as set forth in SEQ ID NO: 40, and a CDR1 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 39. Theantibody may further be an IgG1 or an IgG4 isotype.

In one embodiment, the present invention is directed to an antibody, oran antigen binding fragment thereof, having antigen binding regions ofantibody L3A10. In one embodiment, the invention provides an antibody,or antigen-binding fragment thereof, comprising a heavy chain variabledomain sequence as set forth in SEQ ID NO: 10, and a light chainvariable domain sequence as set forth in SEQ ID NO: 11. In oneembodiment, the invention is directed to an antibody having a heavychain variable domain comprising the CDRs of SEQ ID NO: 10, and a lightchain variable domain comprising the CDRs of SEQ ID NO: 11. In oneembodiment, the invention features an isolated human antibody, orantigen-binding fragment thereof, that comprises a heavy chain variableregion having an amino acid sequence that is at least 95% identical, atleast 96% identical, at least 97% identical, at least 98% identical, orat least 99% identical to the sequence set forth in SEQ ID NO: 10, andcomprises a light chain variable region having an amino acid sequencethat is at least 95% identical, at least 96% identical, at least 97%identical, at least 98% identical, or at least 99% identical to thesequence set forth in SEQ ID NO: 11. In one embodiment, the inventionfeatures an anti-LAG3 antibody, or an antigen-binding portion thereof,comprising a heavy chain variable region comprising a CDR3 domaincomprising the amino acid as set forth in SEQ ID NO: 44, a CDR2 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 43, and aCDR1 domain comprising the amino acid sequence as set forth in SEQ IDNO: 42; and comprising a light chain variable region comprising a CDR3domain comprising the amino acid as set forth in SEQ ID NO: 47, a CDR2domain comprising the amino acid sequence as set forth in SEQ ID NO: 46,and a CDR1 domain comprising the amino acid sequence as set forth in SEQID NO: 45. The antibody may further be an IgG1 or an IgG4 isotype.

In one embodiment, the present invention is directed to an antibody, oran antigen binding fragment thereof, having antigen binding regions ofantibody L3C5. In one embodiment, the invention provides an antibody, orantigen-binding fragment thereof, comprising a heavy chain variabledomain sequence as set forth in SEQ ID NO: 12, and a light chainvariable domain sequence as set forth in SEQ ID NO: 13. In oneembodiment, the invention is directed to an antibody having a heavychain variable domain comprising the CDRs of SEQ ID NO: 12, and a lightchain variable domain comprising the CDRs of SEQ ID NO: 13. In oneembodiment, the invention features an isolated human antibody, orantigen-binding fragment thereof, that comprises a heavy chain variableregion having an amino acid sequence that is at least 95% identical, atleast 96% identical, at least 97% identical, at least 98% identical, orat least 99% identical to the sequence set forth in SEQ ID NO: 12, andcomprises a light chain variable region having an amino acid sequencethat is at least 95% identical, at least 96% identical, at least 97%identical, at least 98% identical, or at least 99% identical to thesequence set forth in SEQ ID NO: 13. In one embodiment, the inventionfeatures an anti-LAG3 antibody, or an antigen-binding portion thereof,comprising a heavy chain variable region comprising a CDR3 domaincomprising the amino acid as set forth in SEQ ID NO: 50, a CDR2 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 49, and aCDR1 domain comprising the amino acid sequence as set forth in SEQ IDNO: 48; and comprising a light chain variable region comprising a CDR3domain comprising the amino acid as set forth in SEQ ID NO: 53, a CDR2domain comprising the amino acid sequence as set forth in SEQ ID NO: 52,and a CDR1 domain comprising the amino acid sequence as set forth in SEQID NO: 51. The antibody may further be an IgG1 or an IgG4 isotype.

In one embodiment, the present invention is directed to an antibody, oran antigen binding fragment thereof, having antigen binding regions ofantibody L3E3. In one embodiment, the invention provides an antibody, orantigen-binding fragment thereof, comprising a heavy chain variabledomain sequence as set forth in SEQ ID NO: 8, and a light chain variabledomain sequence as set forth in SEQ ID NO: 14. In one embodiment, theinvention is directed to an antibody having a heavy chain variabledomain comprising the CDRs of SEQ ID NO: 8, and a light chain variabledomain comprising the CDRs of SEQ ID NO: 14. In one embodiment, theinvention features an isolated human antibody, or antigen-bindingfragment thereof, that comprises a heavy chain variable region having anamino acid sequence that is at least 95% identical, at least 96%identical, at least 97% identical, at least 98% identical, or at least99% identical to the sequence set forth in SEQ ID NO: 8, and comprises alight chain variable region having an amino acid sequence that is atleast 95% identical, at least 96% identical, at least 97% identical, atleast 98% identical, or at least 99% identical to the sequence set forthin SEQ ID NO. 14. In one embodiment, the invention features an anti-LAG3antibody, or an antigen-binding portion thereof, comprising a heavychain variable region comprising a CDR3 domain comprising the amino acidas set forth in SEQ ID NO: 38, a CDR2 domain comprising the amino acidsequence as set forth in SEQ ID NO: 37, and a CDR1 domain comprising theamino acid sequence as set forth in SEQ ID NO: 36; and comprising alight chain variable region comprising a CDR3 domain comprising theamino acid as set forth in SEQ ID NO: 56, a CDR2 domain comprising theamino acid sequence as set forth in SEQ ID NO: 55, and a CDR1 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 54. Theantibody may further be an IgG1 or an IgG4 isotype.

As described in Table 3, antibodies L35D4, L35G6, L33H11, L32A9, L32D10and L32A4, have a heavy chain variable region having an amino acidsequence as set forth in SEQ ID NO.1. As also described in Table 3, SEQID NO.1 is at least 95% identical to SEQ ID NO:8 (as described for L3A1and L3E3).

As described in Table 3, SEQ ID NO: 7 (as described for L32A4) is atleast 95% identical to SEQ ID NO.9 (as described for L3A1).

Antigen-binding fragments of antigen binding proteins of the inventionmay be produced by conventional techniques. Examples of such fragmentsinclude, but are not limited to, Fab and F(ab′)2 fragments.

Single chain antibodies may be formed by linking heavy and light chainvariable domain (Fv region) fragments via an amino acid bridge (shortpeptide linker), resulting in a single polypeptide chain. Suchsingle-chain Fvs (scFvs) have been prepared by fusing DNA encoding apeptide linker between DNAs encoding the two variable domainpolypeptides (VL and VH). The resulting polypeptides can fold back onthemselves to form antigen-binding monomers, or they can form multimers(e.g., dimers, trimers, or tetramers), depending on the length of aflexible linker between the two variable domains (Kortt et al., 1997,Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng. 18:95-108). Bycombining different VL and VH-comprising polypeptides, one can formmultimeric scFvs that bind to different epitopes (Kriangkum et al.,2001, Biomol. Eng. 18:31-40). Techniques developed for the production ofsingle chain antibodies include those described in U.S. Pat. No.4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544, de Graaf etal., 2002, Methods Mol. Biol. 178:379-87.

In certain embodiments, the present disclosure provides a Fab fullyhuman antibody fragment, having a variable domain region from a heavychain and a variable domain region from a light chain, wherein the heavychain variable domain sequence that is at least 95% identical, at least96% identical, at least 97% identical, at least 98% identical, at least99%, or 100% identical, to the amino acid sequences selected from thegroup consisting of SEQ ID NO. 1, SEQ ID NO. 8, SEQ ID NO. 10, SEQ IDNO. 12, and combinations thereof, and that has a light chain variabledomain sequence that is at least 95% identical, at least 96% identical,at least 97% identical, at least 98% identical, at least 99%, or 100%identical to the amino acid sequence consisting of SEQ ID NO. 2, SEQ IDNO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ IDNO: 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, and combinationsthereof. Preferably, the fully human antibody Fab fragment has both aheavy chain variable domain region and a light chain variable domainregion wherein the antibody has a heavy chain/light chain variabledomain sequence selected from the group consisting of SEQ ID NO. 1/SEQID NO. 2, SEQ ID NO. 1/SEQ ID NO. 3, SEQ ID NO. 1/SEQ ID NO. 4, SEQ IDNO. 1/SEQ ID NO. 5, SEQ ID NO. 1/SEQ ID NO. 6, SEQ ID NO. 1/SEQ ID NO.7, SEQ ID NO. 8/SEQ ID NO. 9, SEQ ID NO. 10/SEQ ID NO. 11, SEQ ID NO.12/SEQ ID NO. 13, SEQ ID NO. 8/SEQ ID NO. 14, and combinations thereof.

In one embodiment, the present disclosure provides a single chain humanantibody, having a variable domain region from a heavy chain and avariable domain region from a light chain and a peptide linkerconnection the heavy chain and light chain variable domain regions,wherein the heavy chain variable domain sequence that is at least 95%identical, at least 96% identical, at least 97% identical, at least 98%identical, at least 99% identical, or 100% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.8, SEQ ID NO. 10, SEQ ID NO. 12, and that has a light chain variabledomain sequence that is at least 95% identical, at least 96% identical,at least 97% identical, at least 98% identical, at least 99%, or 100%identical to the amino acid sequence consisting of SEQ ID NO. 2, SEQ IDNO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ IDNO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, and combinationsthereof. Preferably, the fully human single chain antibody has both aheavy chain variable domain region and a light chain variable domainregion, wherein the single chain fully human antibody has a heavychain/light chain variable domain sequence selected from the groupconsisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 1/SEQ ID NO. 3, SEQID NO. 1/SEQ ID NO. 4, SEQ ID NO. 1/SEQ ID NO. 5, SEQ ID NO. 1/SEQ IDNO. 6, SEQ ID NO. 1/SEQ ID NO. 7, SEQ ID NO. 8/SEQ ID NO. 9, SEQ ID NO.10/SEQ ID NO. 11, SEQ ID NO. 12/SEQ ID NO. 13, SEQ ID NO. 8/SEQ ID NO.14, and combinations thereof.

Techniques are known for deriving an antibody of a different subclass orisotype from an antibody of interest, i.e., subclass switching. Thus,IgG antibodies may be derived from an IgM antibody, for example, andvice versa. Such techniques allow the preparation of new antibodies thatpossess the antigen-binding properties of a given antibody (the parentantibody), but also exhibit biological properties associated with anantibody isotype or subclass different from that of the parent antibody.Recombinant DNA techniques may be employed. Cloned DNA encodingparticular antibody polypeptides may be employed in such procedures,e.g., DNA encoding the constant domain of an antibody of the desiredisotype (Lantto et al., 2002, Methods Mol. Biol. 178:303-16). Moreover,if an IgG4 is desired, it may also be desired to introduce a pointmutation (CPSCP→CPPCP) in the hinge region (Bloom et al., 1997, ProteinScience 6:407) to alleviate a tendency to form intra-H chain disulfidebonds that can lead to heterogeneity in the IgG4 antibodies. Thus, inone embodiment, the antibody of the invention is a human IgG1 antibody.Thus, in one embodiment, the antibody of the invention is a human IgG4antibody.

The present disclosure provides a number of antibodies structurallycharacterized by the amino acid sequences of their variable domainregions. However, the amino acid sequences can undergo some changeswhile retaining their high degree of binding to their specific targets.More specifically, many amino acids in the variable domain region can bechanged with conservative substitutions and it is predictable that thebinding characteristics of the resulting antibody will not differ fromthe binding characteristics of the wild type antibody sequence. Thereare many amino acids in an antibody variable domain that do not directlyinteract with the antigen or impact antigen binding and are not criticalfor determining antibody structure. For example, a predictednonessential amino acid residue in any of the disclosed antibodies ispreferably replaced with another amino acid residue from the same class.Methods of identifying amino acid conservative substitutions which donot eliminate antigen binding are well-known in the art (see, e.g.,Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. ProteinEng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA94:412-417 (1997)). Near et al. Mol. Immunol. 30:369-377, 1993 explainshow to impact or not impact binding through site-directed mutagenesis.Near et al. only mutated residues that they thought had a highprobability of changing antigen binding. Most had a modest or negativeeffect on binding affinity (Near et al. Table 3) and binding todifferent forms of digoxin (Near et al. Table 2).

In certain embodiments, an antibody, or antigen-binding fragmentthereof, of the invention has a dissociation constant (K_(D)) of 1×10⁻⁶M or less; 5×10⁻⁷ M or less' 1×10⁻⁷ M or less; 5×10⁻⁸ M or less; 1×10⁻⁸M or less; 5×10⁻⁹ M or less; or 1×10⁻⁹ M or less. In one embodiment, theantibody, or antigen-binding fragment thereof, of the invention as aK_(D) from 1×10⁻⁷ M to 1×10⁻¹⁰ M. In one embodiment, the antibody, orantigen-binding fragment thereof, of the invention as a K_(D) from1×10⁻⁸ M to 1×10⁻¹⁰ M.

Those of ordinary skill in the art will appreciate standard methodsknown for determining the K_(D) of an antibody, or fragment thereof. Forexample, in one embodiment, K_(D) is measured by a radiolabeled antigenbinding assay (RIA). In one embodiment, an RIA is performed with the Fabversion of an antibody of interest and its antigen. For example,solution binding affinity of Fabs for antigen is measured byequilibrating Fab with a minimal concentration of (¹²⁵I)-labeled antigenin the presence of a titration series of unlabeled antigen, thencapturing bound antigen with an anti-Fab antibody-coated plate (see,e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)).

According to another embodiment, K_(D) is measured using a BIACOREsurface plasmon resonance assay. The term “surface plasmon resonance”,as used herein, refers to an optical phenomenon that allows for theanalysis of real-time interactions by detection of alterations inprotein concentrations within a biosensor matrix, for example using theBIACORE system (Biacore Life Sciences division of GE Healthcare,Piscataway, N.J.). Surface plasmon resonance can also be used todetermine K_(off) and K_(a) values.

In particular embodiments, antigen binding proteins of the presentinvention have a binding affinity (K_(a)) for LAG3 of at least 10³M⁻¹S⁻¹. In other embodiments, the antigen binding proteins exhibit aK_(a) of at least 10³ M⁻¹S⁻¹, at least 10⁴ M⁻¹S⁻¹, at least 10⁵ M⁻¹S⁻¹,or at least 10⁶ M⁻¹S⁻¹. In other further embodiments, the antigenbinding proteins exhibit a K_(a) of at least 10⁷ M⁻¹S⁻¹. In otherfurther embodiments, the antigen binding proteins exhibit a K_(a) of atleast 10⁷ M⁻¹S⁻¹, at least 10⁸ M⁻¹S⁻¹, at least 10⁹M⁻¹S⁻¹, or at least10¹⁰ M⁻¹S⁻¹. In one embodiment, the anti-LAG3 antibody, or fragmentthereof, of the invention has a K_(a) of at least 10³-10⁷ M⁻¹S⁻¹. Inanother embodiment, the antigen binding protein exhibits a K_(a)substantially the same as that of an antibody described herein in theExamples. K_(a) can be determined by Biacore testing, for example withBiacore 3000 or T200.

In another embodiment, the present disclosure provides an antigenbinding protein that has a low dissociation rate from LAG3. In oneembodiment, the antigen binding protein has a K_(off) of 1×10⁻⁴ to 10⁻¹sec⁻¹ or lower. In another embodiment, the K_(off) is 5×10⁻⁵ to 10⁻¹sec⁻¹ sec or lower. In another embodiment, the K_(off) is 5×10⁻⁶ to 10⁻¹sec⁻¹ or lower. In another embodiment, the K_(off) is substantially thesame as an antibody described herein. In another embodiment, the antigenbinding protein binds to LAG3 with substantially the same K_(off) as anantibody described herein.

In another aspect, the present disclosure provides an antigen bindingprotein that inhibits an activity of LAG3. In one embodiment, theantigen binding protein has an IC₅₀ of 1000 nM or lower. In anotherembodiment, the IC₅₀ is 100 nM or lower; in another embodiment, the IC₅₀is 10 nM or lower. In another embodiment, the IC₅₀ is substantially thesame as that of an antibody described herein in the Examples. In anotherembodiment, the antigen binding protein inhibits an activity of LAG3with substantially the same IC₅₀ as an antibody described herein.

In another aspect, the present disclosure provides an antigen bindingprotein that binds to LAG3 expressed on the surface of a cell and, whenso bound, inhibits LAG3 signaling activity in the cell without causing asignificant reduction in the amount of LAG3 on the surface of the cell.Any method for determining or estimating the amount of LAG3 on thesurface and/or in the interior of the cell can be used. In otherembodiments, binding of the antigen binding protein to theLAG3-expressing cell causes less than about 75%, 50%, 40%, 30%, 20%,15%, 10%, 5%, 1%, or 0.1% of the cell-surface LAG3 to be internalized.

In another aspect, the present disclosure provides an antigen bindingprotein having a half-life of at least one day in vitro or in vivo(e.g., when administered to a human subject). In one embodiment, theantigen binding protein has a half-life of at least three days. Inanother embodiment, the antigen binding protein has a half-life of fourdays or longer. In another embodiment, the antigen binding protein has ahalf-life of eight days or longer. In another embodiment, the antigenbinding protein is derivatized or modified such that it has a longerhalf-life as compared to the underivatized or unmodified antigen bindingprotein. In another embodiment, the antigen binding protein contains oneor more point mutations to increase serum half life, such as describedin WO00/09560, incorporated by reference herein.

The present disclosure further provides multi-specific antigen bindingproteins, for example, bispecific antigen binding protein, e.g., antigenbinding protein that bind to two different epitopes of LAG3, or to anepitope of LAG3 and an epitope of another molecule, via two differentantigen binding sites or regions. Moreover, bispecific antigen bindingprotein as disclosed herein can comprise a LAG3 binding site from one ofthe herein-described antibodies and a second LAG3 binding region fromanother of the herein-described antibodies, including those describedherein by reference to other publications. Alternatively, a bispecificantigen binding protein may comprise an antigen binding site from one ofthe herein described antibodies and a second antigen binding site fromanother LAG3 antibody that is known in the art, or from an antibody thatis prepared by known methods or the methods described herein.

Numerous methods of preparing bispecific antibodies are known in theart. Such methods include the use of hybrid-hybridomas as described byMilstein et al., 1983, Nature 305:537, and chemical coupling of antibodyfragments (Brennan et al., 1985, Science 229:81; Glennie et al., 1987,J. Immunol. 139:2367; U.S. Pat. No. 6,010,902). Moreover, bispecificantibodies can be produced via recombinant means, for example by usingleucine zipper moieties (i.e., from the Fos and Jun proteins, whichpreferentially form heterodimers; Kostelny et al., 1992, J. Immunol.148:1547) or other lock and key interactive domain structures asdescribed in U.S. Pat. No. 5,582,996. Additional useful techniquesinclude those described in U.S. Pat. Nos. 5,959,083; and 5,807,706.

In another aspect, the antigen binding protein comprises a derivative ofan antibody. The derivatized antibody can comprise any molecule orsubstance that imparts a desired property to the antibody, such asincreased half-life in a particular use. The derivatized antibody cancomprise, for example, a detectable (or labeling) moiety (e.g., aradioactive, colorimetric, antigenic or enzymatic molecule, a detectablebead (such as a magnetic or electrodense (e.g., gold) bead), or amolecule that binds to another molecule (e.g., biotin or streptavidin),a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, orpharmaceutically active moiety), or a molecule that increases thesuitability of the antibody for a particular use (e.g., administrationto a subject, such as a human subject, or other in vivo or in vitrouses). Examples of molecules that can be used to derivatize an antibodyinclude albumin (e.g., human serum albumin) and polyethylene glycol(PEG). Albumin-linked and PEGylated derivatives of antibodies can beprepared using techniques well known in the art. In one embodiment, theantibody is conjugated or otherwise linked to transthyretin (TTR) or aTTR variant. The TTR or TTR variant can be chemically modified with, forexample, a chemical selected from the group consisting of dextran,poly(n-vinyl pyurrolidone), polyethylene glycols, propropylene glycolhomopolymers, polypropylene oxide/ethylene oxide co-polymers,polyoxyethylated polyols and polyvinyl alcohols.

Oligomers that contain one or more antigen binding proteins may beemployed as LAG3 antagonists. Oligomers may be in the form ofcovalently-linked or non-covalently-linked dimers, trimers, or higheroligomers. Oligomers comprising two or more antigen binding protein arecontemplated for use, with one example being a homodimer. Otheroligomers include heterodimers, homotrimers, heterotrimers,homotetramers, heterotetramers, etc.

One embodiment is directed to oligomers comprising multiple antigenbinding proteins joined via covalent or non-covalent interactionsbetween peptide moieties fused to the antigen binding proteins. Suchpeptides may be peptide linkers (spacers), or peptides that have theproperty of promoting oligomerization. Leucine zippers and certainpolypeptides derived from antibodies are among the peptides that canpromote oligomerization of antigen binding proteins attached thereto, asdescribed in more detail below.

In particular embodiments, the oligomers comprise from two to fourantigen binding proteins. The antigen binding proteins of the oligomermay be in any form, such as any of the forms described above, e.g.,variants or fragments. Preferably, the oligomers comprise antigenbinding proteins that have LAG3 binding activity.

In one embodiment, an oligomer is prepared using polypeptides derivedfrom immunoglobulins. Preparation of Fusion Proteins Comprising CertainHeterologous Polypeptides Fused to Various Portions of antibody-derivedpolypeptides (including the Fc domain) has been described, e.g., byAshkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88:10535; Byrn etal., 1990, Nature 344:677; and Hollenbaugh et al., 1992 “Construction ofImmunoglobulin Fusion Proteins”, in Current Protocols in Immunology,Suppl. 4, pages 10.19.1-10.19.11.

One embodiment is directed to a dimer comprising two fusion proteinscreated by fusing a LAG3 binding fragment of an anti-LAG3 antibody tothe Fc region of an antibody. The dimer can be made by, for example,inserting a gene fusion encoding the fusion protein into an appropriateexpression vector, expressing the gene fusion in host cells transformedwith the recombinant expression vector, and allowing the expressedfusion protein to assemble much like antibody molecules, whereuponinterchain disulfide bonds form between the Fc moieties to yield thedimer.

Another method for preparing oligomeric antigen binding proteinsinvolves use of a leucine zipper. Leucine zipper domains are peptidesthat promote oligomerization of the proteins in which they are found.Leucine zippers were originally identified in several DNA-bindingproteins (Landschulz et al., 1988, Science 240:1759), and have sincebeen found in a variety of different proteins. Among the known leucinezippers are naturally occurring peptides and derivatives thereof thatdimerize or trimerize. Examples of leucine zipper domains suitable forproducing soluble oligomeric proteins are described in WO 94/10308, andthe leucine zipper derived from lung surfactant protein D (SPD)described in Hoppe et al., 1994, FEBS Letters 344:191. The use of amodified leucine zipper that allows for stable trimerization of aheterologous protein fused thereto is described in Fanslow et al., 1994,Semin. Immunol. 6:267-78. In one approach, recombinant fusion proteinscomprising an anti-LAG3 antibody fragment or derivative fused to aleucine zipper peptide are expressed in suitable host cells, and thesoluble oligomeric anti-LAG3 antibody fragments or derivatives that formare recovered from the culture supernatant.

Antigen binding proteins directed against LAG3 can be used, for example,in assays to detect the presence of LAG3 polypeptides, either in vitroor in vivo. The antigen binding proteins also may be employed inpurifying LAG3 proteins by immunoaffinity chromatography. Blockingantigen binding proteins can be used in the methods disclosed herein.Such antigen binding proteins that function as LAG3 antagonists may beemployed in treating any LAG3-induced condition, including but notlimited to various cancers.

Antigen binding proteins may be employed in an in vitro procedure, oradministered in vivo to inhibit LAG3-induced biological activity.Disorders caused or exacerbated (directly or indirectly) by theproteolytic of LAG3, examples of which are provided herein, thus may betreated. In one embodiment, the present invention provides a therapeuticmethod comprising in vivo administration of a LAG3 blocking antigenbinding protein to a mammal in need thereof in an amount effective forreducing a LAG3-induced biological activity.

In certain embodiments of the invention, antigen binding proteinsinclude fully human monoclonal antibodies that inhibit a biologicalactivity of LAG3.

Antigen binding proteins, including antibodies and antibody fragmentsdescribed herein, may be prepared by any of a number of conventionaltechniques. For example, they may be purified from cells that naturallyexpress them (e.g., an antibody can be purified from a hybridoma thatproduces it), or produced in recombinant expression systems, using anytechnique known in the art. See, for example, Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Kennet et al.(eds.), Plenum Press, New York (1980); and Antibodies: A LaboratoryManual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., (1988).

Any expression system known in the art can be used to make therecombinant polypeptides, including antibodies and antibody fragmentsdescribed herein, of the invention. In general, host cells aretransformed with a recombinant expression vector that comprises DNAencoding a desired polypeptide. Among the host cells that may beemployed are prokaryotes, yeast or higher eukaryotic cells. Prokaryotesinclude gram negative or gram positive organisms, for example E. coli orbacilli. Higher eukaryotic cells include insect cells and establishedcell lines of mammalian origin. Examples of suitable mammalian host celllines include the COS-7 line of monkey kidney cells (ATCC CRL 1651)(Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK(ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived from theAfrican green monkey kidney cell line CV1 (ATCC CCL 70) as described byMcMahan et al., 1991, EMBO J. 10: 2821. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described by Pouwels et al. (Cloning Vectors: ALaboratory Manual, Elsevier, N.Y., 1985).

The transformed cells can be cultured under conditions that promoteexpression of the polypeptide, and the polypeptide recovered byconventional protein purification procedures. One such purificationprocedure includes the use of affinity chromatography, e.g., over amatrix having all or a portion (e.g., the extracellular domain) of LAG3bound thereto. Polypeptides contemplated for use herein includesubstantially homogeneous recombinant mammalian anti-LAG3 antibodypolypeptides substantially free of contaminating endogenous materials.

Antigen binding proteins may be prepared, and screened for desiredproperties, by any of a number of known techniques. Certain of thetechniques involve isolating a nucleic acid encoding a polypeptide chain(or portion thereof) of an antigen binding protein of interest (e.g., ananti-LAG3 antibody), and manipulating the nucleic acid throughrecombinant DNA technology. The nucleic acid may be fused to anothernucleic acid of interest, or altered (e.g., by mutagenesis or otherconventional techniques) to add, delete, or substitute one or more aminoacid residues, for example.

Polypeptides of the present disclosure can be produced using anystandard methods known in the art. In one example, the polypeptides areproduced by recombinant DNA methods by inserting a nucleic acid sequence(a cDNA) encoding the polypeptide into a recombinant expression vectorand expressing the DNA sequence under conditions promoting expression.The invention includes nucleic acids encoding any of the polypeptidesequences described in SEQ ID Nos: 1 to 56, as well as vectorscomprising said nucleic acid sequences.

Nucleic acids encoding any of the various polypeptides disclosed hereinmay be synthesized chemically. Codon usage may be selected so as toimprove expression in a cell. Such codon usage will depend on the celltype selected. Specialized codon usage patterns have been developed forE. coli and other bacteria, as well as mammalian cells, plant cells,yeast cells and insect cells.

General techniques for nucleic acid manipulation are described forexample in Sambrook et al., Molecular Cloning: A Laboratory Manual,Vols. 1-3, Cold Spring Harbor Laboratory Press, 2 ed., 1989, or F.Ausubel et al., Current Protocols in Molecular Biology (Green Publishingand Wiley-Interscience: New York, 1987) and periodic updates, hereinincorporated by reference. The DNA encoding the polypeptide is operablylinked to suitable transcriptional or translational regulatory elementsderived from mammalian, viral, or insect genes. Such regulatory elementsinclude a transcriptional promoter, an optional operator sequence tocontrol transcription, a sequence encoding suitable mRNA ribosomalbinding sites, and sequences that control the termination oftranscription and translation. The ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants is additionally incorporated.

The recombinant DNA can also include any type of protein tag sequencethat may be useful for purifying the protein. Examples of protein tagsinclude but are not limited to a histidine tag, a FLAG tag, a myc tag,an HA tag, or a GST tag. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts can befound in Cloning Vectors: A Laboratory Manual, (Elsevier, N.Y., 1985).

The expression construct is introduced into the host cell using a methodappropriate to the host cell. A variety of methods for introducingnucleic acids into host cells are known in the art, including, but notlimited to, electroporation; transfection employing calcium chloride,rubidium chloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (where thevector is an infectious agent). Suitable host cells include prokaryotes,yeast, mammalian cells, or bacterial cells.

Suitable bacteria include gram negative or gram positive organisms, forexample, E. coli or Bacillus spp. Yeast, preferably from theSaccharomyces species, such as S. cerevisiae, may also be used forproduction of polypeptides. Various mammalian or insect cell culturesystems can also be employed to express recombinant proteins.Baculovirus systems for production of heterologous proteins in insectcells are reviewed by Luckow and Summers, (Bio/Technology, 6:47, 1988).Examples of suitable mammalian host cell lines include endothelialcells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3, Chinesehamster ovary (CHO), human embryonic kidney cells, HeLa, 293, 293T, andBHK cell lines. Purified polypeptides are prepared by culturing suitablehost/vector systems to express the recombinant proteins. For manyapplications, the small size of many of the polypeptides disclosedherein would make expression in E. coli as the preferred method forexpression. The protein is then purified from culture media or cellextracts.

Proteins can also be produced using cell-translation systems. For suchpurposes the nucleic acids encoding the polypeptide must be modified toallow in vitro transcription to produce mRNA and to allow cell-freetranslation of the mRNA in the particular cell-free system beingutilized (eukaryotic such as a mammalian or yeast cell-free translationsystem or prokaryotic such as a bacterial cell-free translation system.

LAG3-binding polypeptides can also be produced by chemical synthesis(such as by the methods described in Solid Phase Peptide Synthesis, 2nded., 1984, The Pierce Chemical Co., Rockford, Ill.). Modifications tothe protein can also be produced by chemical synthesis.

The polypeptides of the present disclosure can be purified byisolation/purification methods for proteins generally known in the fieldof protein chemistry. Non-limiting examples include extraction,recrystallization, salting out (e.g., with ammonium sulfate or sodiumsulfate), centrifugation, dialysis, ultrafiltration, adsorptionchromatography, ion exchange chromatography, hydrophobic chromatography,normal phase chromatography, reversed-phase chromatography, gelfiltration, gel permeation chromatography, affinity chromatography,electrophoresis, countercurrent distribution or any combinations ofthese. After purification, polypeptides may be exchanged into differentbuffers and/or concentrated by any of a variety of methods known to theart, including, but not limited to, filtration and dialysis.

The purified polypeptide is preferably at least 85% pure, morepreferably at least 95% pure, and most preferably at least 98% pure.Regardless of the exact numerical value of the purity, the polypeptideis sufficiently pure for use as a pharmaceutical product.

In certain embodiments, the present disclosure provides monoclonalantibodies that bind to LAG3. Monoclonal antibodies may be producedusing any technique known in the art, e.g., by immortalizing spleencells harvested from the transgenic animal after completion of theimmunization schedule. The spleen cells can be immortalized using anytechnique known in the art, e.g., by fusing them with myeloma cells toproduce hybridomas. Myeloma cells for use in hybridoma-producing fusionprocedures preferably are non-antibody-producing, have high fusionefficiency, and enzyme deficiencies that render them incapable ofgrowing in certain selective media which support the growth of only thedesired fused cells (hybridomas). Examples of suitable cell lines foruse in mouse fusions include Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul;examples of cell lines used in rat fusions include R210.RCY3, Y3-Ag1.2.3, IR983F and 48210. Other cell lines useful for cell fusions areU-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6.

Antigen-binding fragments of antigen binding proteins of the inventionmay be produced by conventional techniques.

Post-Translational Modifications of Polypeptides

In certain embodiments, the binding polypeptides of the invention mayfurther comprise post-translational modifications. Exemplarypost-translational protein modifications include phosphorylation,acetylation, methylation, ADP-ribosylation, ubiquitination,glycosylation, carbonylation, sumoylation, biotinylation or addition ofa polypeptide side chain or of a hydrophobic group. As a result, themodified soluble polypeptides may contain non-amino acid elements, suchas lipids, poly- or mono-saccharide, and phosphates. A preferred form ofglycosylation is sialylation, which conjugates one or more sialic acidmoieties to the polypeptide. Sialic acid moieties improve solubility andserum half-life while also reducing the possible immunogeneticity of theprotein. See Raju et al. Biochemistry. 2001 31; 40(30):8868-76.

In one embodiment, modified forms of the subject soluble polypeptidescomprise linking the subject soluble polypeptides to nonproteinaceouspolymers. In one embodiment, the polymer is polyethylene glycol (“PEG”),polypropylene glycol, or polyoxyalkylenes, in the manner as set forth inU.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337.

PEG is a water soluble polymer that is commercially available or can beprepared by ring-opening polymerization of ethylene glycol according tomethods well known in the art (Sandler and Karo, Polymer Synthesis,Academic Press, New York, Vol. 3, pages 1123-161). The term “PEG” isused broadly to encompass any polyethylene glycol molecule, withoutregard to size or to modification at an end of the PEG, and can berepresented by the formula: X—O(CH₂CH₂O)_(n)—CH₂CH₂OH (1), where n is 20to 2300 and X is H or a terminal modification, e.g., a C₁₋₄ alkyl. Inone embodiment, the PEG of the invention terminates on one end withhydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”). A PEG cancontain further chemical groups which are necessary for bindingreactions; which results from the chemical synthesis of the molecule; orwhich is a spacer for optimal distance of parts of the molecule. Inaddition, such a PEG can consist of one or more PEG side-chains whichare linked together. PEGs with more than one PEG chain are calledmultiarmed or branched PEGs. Branched PEGs can be prepared, for example,by the addition of polyethylene oxide to various polyols, includingglycerol, pentaerythriol, and sorbitol. For example, a four-armedbranched PEG can be prepared from pentaerythriol and ethylene oxide.Branched PEG are described in, for example, EP-A 0 473 084 and U.S. Pat.No. 5,932,462. One form of PEGs includes two PEG side-chains (PEG2)linked via the primary amino groups of a lysine (Monfardini et al.,Bioconjugate Chem. 6 (1995) 62-69).

The serum clearance rate of PEG-modified polypeptide may be decreased byabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or even 90%, relative tothe clearance rate of the unmodified binding polypeptide. ThePEG-modified polypeptide may have a half-life (t_(1/2)) which isenhanced relative to the half-life of the unmodified protein. Thehalf-life of PEG-binding polypeptide may be enhanced by at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%,250%, 300%, 400% or 500%, or even by 1000% relative to the half-life ofthe unmodified binding polypeptide. In some embodiments, the proteinhalf-life is determined in vitro, such as in a buffered saline solutionor in serum. In other embodiments, the protein half-life is an in vivohalf-life, such as the half-life of the protein in the serum or otherbodily fluid of an animal.

Therapeutic Methods, Formulations and Modes of Administration

The present disclosure further provides a method for treating a broadspectrum of mammalian cancers, infectious diseases, or autoimmunereactions,

In one embodiment, the present disclosure features methods for treatingor preventing the S. aureus infection comprising administering anti-LAG3antibodies or antigen binding fragments of the present invention.

The present disclosure further provides a method for treating a broadspectrum of mammalian cancers, infectious diseases, or autoimmunereactions, comprising administering an anti-LAG3 polypeptide using theantibodies, and antibody fragments, disclosed herein. In one embodiment,the invention provides a method of treating cancer by administering ananti-human LAG3 antibody to a subject in need thereof. Examples ofantibodies, and fragments thereof, that may be used in the therapeuticsmethods disclosed herein include an anti-human LAG3 human antibody of anIgG class having a binding affinity of at least 10⁻⁶ M, or an anti-humanLAG3 Fab antibody fragment comprising a heavy chain variable region anda light chain variable region from the antibody sequences described inSEQ ID Nos. 1-14 or comprising the CDRs described in any of the antibodysequences of SEQ ID Nos: 1-14. In one embodiment, the methods disclosedherein comprise administering a fully human antibody comprising a heavychain variable domain sequence that is at least 95% identical, at least96% identical, at least 97% identical, at least 98% identical, or atleast 99% identical, to an amino acid sequence selected from the groupconsisting of SEQ ID NO. 1, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12,and combinations thereof, and having a light chain variable domainsequence that is at least 95% identical, at least 96% identical, atleast 97% identical, at least 98% identical, or at least 99% identical,to an amino acid sequence selected from the group consisting of SEQ IDNO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ IDNO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, andcombinations thereof. In one embodiment, the methods disclosed hereincomprise administering an IgG human anti-hLAG3 antibody comprising aheavy chain variable domain sequence selected from the group consistingof SEQ ID NO. 1, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, and havinga light chain variable domain sequence selected form the groupconsisting of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5,SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13,SEQ ID NO. 14.

In one embodiment, the methods described herein include the use of afully human Fab antibody fragment comprising a heavy chain variabledomain sequence that is at least 95% identical, at least 96% identical,at least 97% identical, at least 98% identical, or at least 99%identical, to an amino acid sequence selected from the group consistingof SEQ ID NO. 1, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, andcombinations thereof, and comprising a light chain variable domainsequence that is at least 95% identical, at least 96% identical, atleast 97% identical, at least 98% identical, or at least 99% identical,to an amino acid sequence selected from the group consisting of SEQ IDNO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ IDNO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, andcombinations thereof. In one embodiment, the methods described hereininclude the use of a human Fab antibody fragment comprising a heavychain variable domain sequence selected from the group consisting of SEQID NO. 1, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, and comprising alight chain variable domain sequence selected from the group consistingof SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6,SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14.

In one embodiment, the methods described herein include the use of asingle chain human antibody comprising a heavy chain variable domainsequence that is at least 95% identical, at least 96% identical, atleast 97% identical, at least 98% identical, or at least 99% identical,to an amino acid sequence selected from the group consisting of SEQ IDNO. 1, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, and combinationsthereof, and comprising a light chain variable domain sequence that isat least 95% identical, at least 96% identical, at least 97% identical,at least 98% identical, or at least 99% identical, to an amino acidsequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO.3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9,SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, and combinations thereof.In one embodiment, the methods described herein include the use of asingle chain human antibody comprising a heavy chain variable domainhaving an amino acid sequence selected from the group consisting of SEQID NO. 1, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, and comprising alight chain variable domain having an amino acid sequence selected fromthe group consisting of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ IDNO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ IDNO. 13, SEQ ID NO. 14

In one embodiment, the fully human antibody has both a heavy chain and alight chain wherein the antibody has a heavy chain/light chain variabledomain sequence selected from the group consisting of SEQ ID NO. 1/SEQID NO. 2 (called L35D4 herein), SEQ ID NO. 1/SEQ ID NO. 3 (called L35G6herein), SEQ ID NO. 1/SEQ ID NO. 4 (called L33H11 herein), SEQ ID NO.1/SEQ ID NO. 5 (called L32A9 herein), SEQ ID NO. 1/SEQ ID NO. 6 (calledL32D10 herein), SEQ ID NO. 1/SEQ ID NO. 7 (called L32A4 herein), SEQ IDNO. 8/SEQ ID NO. 9 (called L3A1 herein), SEQ ID NO. 10/SEQ ID NO. 11(called L3A10 herein), SEQ ID NO. 12/SEQ ID NO. 13 (called L3C5 herein),SEQ ID NO. 8/SEQ ID NO. 14 (called L3E3 herein), and combinationsthereof.

In one embodiment, a fully human antibody Fab fragment has both a heavychain variable domain region and a light chain variable domain regionwherein the antibody has a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2(called L35D4 herein), SEQ ID NO. 1/SEQ ID NO. 3 (called L35G6 herein),SEQ ID NO. 1/SEQ ID NO. 4 (called L33H11 herein), SEQ ID NO. 1/SEQ IDNO. 5 (called L32A9 herein), SEQ ID NO. 1/SEQ ID NO. 6 (called L32D10herein), SEQ ID NO. 1/SEQ ID NO. 7 (called L32A4 herein), SEQ ID NO.8/SEQ ID NO. 9 (called L3A1 herein), SEQ ID NO. 10/SEQ ID NO. 11 (calledL3A10 herein), SEQ ID NO. 12/SEQ ID NO. 13 (called L3C5 herein), SEQ IDNO. 8/SEQ ID NO. 14 (called L3E3 herein).

In one embodiment, a fully human single chain antibody has both a heavychain variable domain region and a light chain variable domain region,wherein the single chain fully human antibody has a heavy chain/lightchain variable domain sequence selected from the group consisting of SEQID NO. 1/SEQ ID NO. 2, SEQ ID NO. 1/SEQ ID NO. 3, SEQ ID NO. 1/SEQ IDNO. 4, SEQ ID NO. 1/SEQ ID NO. 5, SEQ ID NO. 1/SEQ ID NO. 6, SEQ ID NO.1/SEQ ID NO. 7, SEQ ID NO. 8/SEQ ID NO. 9, SEQ ID NO. 10/SEQ ID NO. 11,SEQ ID NO. 12/SEQ ID NO. 13, SEQ ID NO. 8/SEQ ID NO. 14.

Cancer Indications

Anti-LAG3 antibodies and antibody fragments of the invention may be usedto treat cancer. Examples of cancer that may be treated include, but arenot limited to, glioblastoma, non-Hodgkin's lymphoma (NHL), Burkitt'slymphoma (BL), multiple myeloma (MM), B chronic lymphocytic leukemia(B-CLL), B and T acute lymphocytic leukemia (ALL), T cell lymphoma(TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL),Hodgkin's Lymphoma (HL), and chronic myeloid leukemia (CML).

In one embodiment, the LAG3 antibodies and antibody fragments describedherein are useful in treating, delaying the progression of, preventingrelapse of or alleviating a symptom of a cancer or other neoplasticcondition, including, hematological malignancies and/or LAG3+ tumors.The LAG3 antibodies and antibody fragments described herein are usefulin treating a cancer selected from the group consisting of non-Hodgkin'slymphoma (NHL), acute lymphocytic leukemia (ALL), acute myeloid leukemia(AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia(CML), multiple myeloma (MM), breast cancer, ovarian cancer, head andneck cancer, bladder cancer, melanoma, colorectal cancer, pancreaticcancer, lung cancer, leiomyoma, leiomyosarcoma, glioma, glioblastoma,and solid tumors, wherein solid tumors are selected from the groupconsisting of breast tumors, ovarian tumors, lung tumors, pancreatictumors, prostate tumors, melanoma tumors, colorectal tumors, lungtumors, head and neck tumors, bladder tumors, esophageal tumors, livertumors, and kidney tumors.

As used herein, “hematological cancer” refers to a cancer of the blood,and includes leukemia, lymphoma and myeloma among others. “Leukemia”refers to a cancer of the blood in which too many white blood cells thatare ineffective in fighting infection are made, thus crowding out theother parts that make up the blood, such as platelets and red bloodcells. Cases of leukemia are classified as acute or chronic.

Certain forms of leukemia include, acute lymphocytic leukemia (ALL);acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL);chronic myelogenous leukemia (CML); Myeloproliferative disorder/neoplasm(MPDS); and myelodysplasia syndrome. “Lymphoma” may refer to a Hodgkin'slymphoma, both indolent and aggressive non-Hodgkin's lymphoma, Burkitt'slymphoma, and follicular lymphoma (small cell and large cell), amongothers. Myeloma may refer to multiple myeloma (MM), giant cell myeloma,heavy-chain myeloma, and light chain or Bence-Jones myeloma.

Blockade of LAG3 by antibodies can enhance an immune response againstcancerous cells in the patient. An anti-LAG3 antibody or antibodyfragment disclosed herein can be used alone to inhibit the growth ofcancerous tumors. Alternatively, an anti-LAG3 antibody or antibodyfragment disclosed herein can be used in conjunction with otherimmunogenic agents, standard cancer treatments, or other antibodies. Inone embodiment, the present disclosure provides a method of inhibitinggrowth of tumor cells in a subject, comprising administering to thesubject a therapeutically effective amount of an anti-LAG3 antibody, orantigen-binding fragment thereof. Preferably, the antibody or antibodyfragment is a human anti-LAG-3 antibody or antibody fragment (such asany of the human anti-LAG3 antibodies described herein).

In one embodiment, preferred cancers whose growth may be inhibitedinclude cancers typically responsive to immunotherapy. Non-limitingexamples of preferred cancers for treatment include melanoma (e.g.,metastatic malignant melanoma), renal cancer (e.g. clear cellcarcinoma), prostate cancer (e.g. hormone refractory prostateadenocarcinoma), breast cancer, colon cancer, fibrosarcoma, and lungcancer (e.g. non-small cell lung cancer). Examples of other cancers thatcan be treated using the disclosed antibodies include bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular malignant melanoma, uterine cancer, ovarian cancer, rectalcancer, cancer of the anal region, stomach cancer, testicular cancer,carcinoma of the fallopian tubes, carcinoma of the endometrium,carcinoma of the cervix, carcinoma of the vagina, carcinoma of thevulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, chronic or acute leukemias including acutemyeloid leukemia, chronic myeloid leukemia, acute lymphoblasticleukemia, chronic lymphocytic leukemia, solid tumors of childhood,lymphocytic lymphoma, cancer of the bladder, cancer of the kidney orureter, carcinoma of the renal pelvis, neoplasm of the central nervoussystem (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axistumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma,environmentally induced cancers including those induced by asbestos, andcombinations of said cancers. Other cancers that can be treated with thedisclosed antibodies are metastatic cancers, especially metastaticcancers that express PD-L1 (Iwai et al. (2005) Int. Immunol.17:133-144).

Optionally antibodies and antibody fragments to LAG3 described hereincan be combined with an immunogenic agent, such as cancerous cells,purified tumor antigens (including recombinant proteins, peptides, andcarbohydrate molecules), cells, and cells transfected with genesencoding immune stimulating cytokines (He et al (2004) J. Immunol.173:4919-28). Non-limiting examples of tumor vaccines that can be usedinclude peptides of melanoma antigens, such as peptides of gp100, MAGEantigens, Trp-2, MART1 and/or tyrosinase, or tumor cells transfected toexpress the cytokine GM-CSF (discussed further below).

In humans, some tumors have been shown to be immunogenic such asmelanomas. By raising the threshold of T cell activation by LAG3blockade, the tumor responses in the host can be activated.

LAG3 blockade is likely to be more effective when combined with avaccination protocol. Many experimental strategies for vaccinationagainst tumors have been devised. In one of these strategies, a vaccineis prepared using autologous or allogeneic tumor cells. These cellularvaccines have been shown to be most effective when the tumor cells aretransduced to express GM-CSF. GM-CSF has been shown to be a potentactivator of antigen presentation for tumor vaccination (Dranoff et al.(1993) Proc. Natl. Acad. Sci U.S.A. 90: 3539-43).

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so called tumor specificantigens (Rosenberg, S A (1999) Immunity 10: 281-7). In many cases,these tumor specific antigens are differentiation antigens expressed inthe tumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp100, MAGE antigens, and Trp-2. More importantly,many of these antigens can be shown to be the targets of tumor specificT cells found in the host. LAG3 blockade can be used in conjunction witha collection of recombinant proteins and/or peptides expressed in atumor in order to generate an immune response to these proteins. Theseproteins are normally viewed by the immune system as self antigens andare therefore tolerant to them. The tumor antigen can include theprotein telomerase, which is required for the synthesis of telomeres ofchromosomes and which is expressed in more than 85% of human cancers andin only a limited number of somatic tissues (Kim et al. (1994) Science266: 2011-2013). (These somatic tissues may be protected from immuneattack by various means). Tumor antigen can also be “neo-antigens”expressed in cancer cells because of somatic mutations that alterprotein sequence or create fusion proteins between two unrelatedsequences (i.e., bcr-abl in the Philadelphia chromosome), or idiotypefrom B cell tumors.

Other tumor vaccines can include the proteins from viruses implicated inhuman cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses(HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). Another form oftumor specific antigen which can be used in conjunction with LAG3blockade is purified heat shock proteins (HSP) isolated from the tumortissue itself. These heat shock proteins contain fragments of proteinsfrom the tumor cells and these HSPs are highly efficient at delivery toantigen presenting cells for eliciting tumor immunity (Suot & Srivastava(1995) Science 269:1585-1588; Tamura et al. (1997) Science 278:117-120).

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DC's can be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs canalso be transduced by genetic means to express these tumor antigens aswell. DCs have also been fused directly to tumor cells for the purposesof immunization (Kugler et al. (2000) Nature Medicine 6:332-336). As amethod of vaccination, DC immunization can be effectively combined withLAG3 blockade to activate more potent anti-tumor responses.

LAG3 blockade (using the anti-LAG3 antibodies and fragments disclosedherein) can also be combined with standard cancer treatments. LAG3blockade can be effectively combined with chemotherapeutic regimes. Inthese instances, it may be possible to reduce the dose ofchemotherapeutic reagent administered (Mokyr et al. (1998) CancerResearch 58: 5301-5304). An example of such a combination is ananti-LAG3 antibody in combination with decarbazine for the treatment ofmelanoma. Another example of such a combination is an anti-LAG3 antibodyin combination with interleukin-2 (IL-2) for the treatment of melanoma.The scientific rationale behind the combined use of LAG3 blockade andchemotherapy is that cell death, that is a consequence of the cytotoxicaction of most chemotherapeutic compounds, should result in increasedlevels of tumor antigen in the antigen presentation pathway. Othercombination therapies that may result in synergy with LAG3 blockadethrough cell death are radiation, surgery, and hormone deprivation. Eachof these protocols creates a source of tumor antigen in the host.Angiogenesis inhibitors can also be combined with LAG3 blockadeInhibition of angiogenesis often leads to tumor cell death which mayfeed tumor antigens into host antigen presentation pathways.

Bispecific antibodies can be used to target two separate antigens. Forexample anti-Fc receptor/anti-tumor antigen (e.g., Her-2/neu) bispecificantibodies have been used to target macrophages to sites of tumor. Thistargeting may more effectively activate tumor specific responses. The Tcell arm of these responses would be augmented by the use of LAG3blockade using anti-LAG3 antibodies and antibody fragments describedherein. Alternatively, antigen may be delivered directly to DCs by theuse of bispecific antibodies which bind to tumor antigen and a dendriticcell specific cell surface marker.

Bispecific antibodies can be used to target two separate tumor antigens.A variety of tumor targets may be considered, including, for example,Her2, cMet, EGFR and VEGFR expressing tumors. As such, in oneembodiment, the invention provides a bispecific antibody comprising ananti-LAG3 antibody (or antigen binding fragment) comprising a heavy andlight chain variable region sequence as described herein or a heavy andlight chain variable region comprising a set of CDR sequences asdescribed herein and an anti-Her2, an anti-EGFR, an anti-VEGFR (see, forexamples, antibodies described in U.S. Pat. No. 9,029,510, incorporatedby reference herein), or an anti-cMet antibody (or antigen bindingportion thereof). In one embodiment, the invention includes a bispecificantibody specific to LAG3 and EGFR, wherein the antibody comprises ananti-LAG3 antibody or fragment as disclosed herein and an anti-EGFRantibody or fragment as described in International Publication No. WO2013/173255 or International Publication No. WO 2014/066530, both ofwhich are incorporated by reference in their entireties herein. In oneembodiment, the invention includes a bispecific antibody specific toLAG3 and VEGFR, wherein the antibody comprises an anti-LAG3 antibody orfragment as disclosed herein and an anti-VEGFR antibody or fragment asdescribed in U.S. Pat. No. 9,029,510, incorporated by reference in itsentirety herein. In one embodiment, the invention includes a bispecificantibody specific to LAG3 and cMet, wherein the antibody comprises ananti-LAG3 antibody or fragment as disclosed herein and an anti-cMetantibody or fragment as described in International Publication No. WO2016/094455, incorporated by reference in its entirety herein.

LAG3 blocking antibodies and antibody fragments described herein canalso be used in combination with bispecific antibodies that target, forexample, Fcα or Fcγ receptor-expressing effectors cells to tumor cells(U.S. Pat. Nos. 5,922,845 and 5,837,243).

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation of proteinswhich are expressed by the tumors and which are immunosuppressive. Theseinclude among others TGF-β (Kehrl et al. (1986) J. Exp. Med. 163:1037-1050), IL-10 (Howard & O'Garra (1992) Immunology Today 13:198-200), and Fas ligand (Hahne et al. (1996) Science 274: 1363-1365).Antibodies to each of these entities can be used in combination withanti-LAG3 antibodies and antibody fragments described herein tocounteract the effects of the immunosuppressive agent and favor tumorimmune responses by the host.

Other antibodies which activate host immune responsiveness can be usedin combination with anti-LAG3 antibodies and antibody fragmentsdescribed herein. These include molecules on the surface of dendriticcells which activate DC function and antigen presentation. Anti-CD40antibodies are able to substitute effectively for T cell helper activity(Ridge et al. (1998) Nature 393: 474-478) and can be used in conjunctionwith LAG3 antibodies (Ito et al. (2000) Immunobiology 201 (5) 527-40).Activating antibodies to T cell costimulatory molecules such as CTLA-4,OX-40, 4-1BB, and ICOS may also provide for increased levels of T cellactivation. LAG3 blockade can be used to increase the effectiveness ofthe donor engrafted tumor specific T cells.

There are also several experimental treatment protocols that involve exvivo activation and expansion of antigen specific T cells and adoptivetransfer of these cells into recipients in order to stimulateantigen-specific T cells against tumor (Greenberg & Riddell (1999)Science 285: 546-51). These methods can also be used to activate T cellresponses to infectious agents such as CMV. Ex vivo activation in thepresence of anti-LAG3 antibodies can increase the frequency and activityof the adoptively transferred T cells.

Additional methods for treating cancer using the anti-LAG3 antibodiesand fragments of the invention are disclosed below, for example, in theCombination Therapy section.

Infectious Diseases

The present disclosure further provides a method of treating aninfectious disease in a subject comprising administering to the subjectan anti-LAG3 antibody, or antigen-binding portion thereof, such that thesubject is treated for the infectious disease. Preferably, the antibodyis a human anti-human LAG3 antibody or antibody fragment (such as any ofthe human anti-LAG-3 antibodies described herein) Similar to itsapplication to tumors, antibody mediated LAG3 blockade can be usedalone, or as an adjuvant, in combination with vaccines, to stimulate theimmune response to pathogens, toxins, and self-antigens. Examples ofpathogens for which this therapeutic approach can be particularlyuseful, include pathogens for which there is currently no effectivevaccine, or pathogens for which conventional vaccines are less thancompletely effective. These include, but are not limited to HIV,Hepatitis (A, B, & C), Influenza, Herpes, Giardia, Malaria, Leishmania,Staphylococcus aureus, Pseudomonas aeruginosa. LAG3 blockade isparticularly useful against established infections by agents such as HIVthat present altered antigens over the course of the infections. Thesenovel epitopes are recognized as foreign at the time of anti-human LAG3administration, thus provoking a strong T cell response that is notdampened by negative signals through LAG3.

Some examples of pathogenic viruses causing infections treatable by thedisclosed antibodies include HIV, hepatitis (A, B, or C), herpes virus(e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus),adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus,coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus,rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus,HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus,rabies virus, JC virus and arboviral encephalitis virus.

Some examples of pathogenic bacteria causing infections treatable by thedisclosed antibodies include chlamydia, rickettsial bacteria,mycobacteria, staphylococci, streptococci, pneumonococci, meningococciand gonococci, klebsiella, proteus, serratia, pseudomonas, legionella,diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax,plague, leptospirosis, and Lymes disease bacteria.

Some examples of pathogenic fungi causing infections treatable by thedisclosed antibodies include Candida (albicans, krusei, glabrata,tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus,niger, etc.), Genus Mucorales (mucor, absidia, rhizopus), Sporothrixschenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis,Coccidioides immitis and Histoplasma capsulatum.

Some examples of pathogenic parasites causing infections treatable bythe disclosed antibodies include Entamoeba histolytica, Balantidiumcoli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia,Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesiamicroti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani,Toxoplasma gondii, Nippostrongylus brasiliensis.

LAG3 blockade can be combined with other forms of immunotherapy such ascytokine treatment (e.g., interferons, GM-CSF, G-CSF, IL-2), orbispecific antibody therapy, which provides for enhanced presentation oftumor antigens (see, e.g., Holliger (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak (1994) Structure 2:1121-1123).

Autoimmune Reactions

Anti-LAG3 antibodies may provoke and amplify autoimmune responses.Indeed, induction of anti-tumor responses using tumor cell and peptidevaccines reveals that many anti-tumor responses involve anti-selfreactivities (van Elsas et al. (2001) J. Exp. Med. 194:481-489;Overwijk, et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96: 2982-2987;Hurwitz, (2000) supra; Rosenberg & White (1996) J. Immunother EmphasisTumor Immunol 19 (1): 81-4). Therefore, it is possible to consider usinganti-LAG3 antibodies like those described herein in a LAG3 blockade inconjunction with various self proteins in order to devise vaccinationprotocols to efficiently generate immune responses against these selfproteins for disease treatment. For example, Alzheimer's diseaseinvolves inappropriate accumulation of Aβ peptide in amyloid deposits inthe brain; antibody responses against amyloid are able to clear theseamyloid deposits (Schenk et al., (1999) Nature 400: 173-177).

Other self proteins can also be used as targets such as IgE for thetreatment of allergy and asthma, and TNFα for rheumatoid arthritis.Finally, antibody responses to various hormones may be induced by theuse of anti-LAG-3 antibody. Neutralizing antibody responses toreproductive hormones can be used for contraception. Neutralizingantibody response to hormones and other soluble factors that arerequired for the growth of particular tumors can also be considered aspossible vaccination targets.

Analogous methods as described above for the use of anti-LAG3 antibodiesand antibody fragments can be used for induction of therapeuticautoimmune responses to treat patients having an inappropriateaccumulation of other self-antigens, such as amyloid deposits, includingAβ in Alzheimer's disease, cytokines such as TNFα, and IgE. Further,anti-LAG3 antibodies and antibody fragments can be used for induction oftherapeutic autoimmune responses to treat patients having otherautoimmune diseases, including but not limited to, celiac disease,Crohn's disease, Grave's disease, inflammatory bowel disease (IBD),lupus, multiple sclerosis, Myasthenia Gravis, polymyalgia rheumatic,rheumatoid arthritis, type I diabetes, and vasculitis.

Vaccines

The anti-LAG3 antibodies and antibody fragments of the invention can beused to stimulate antigen-specific immune responses by coadministrationof an anti-LAG3 antibody or antibody portion with an antigen of interest(e.g., a vaccine). Accordingly, this disclosure further provides amethod of enhancing an immune response to an antigen in a subject,comprising administering to the subject: (i) the antigen; and (ii) ananti-LAG3 antibody, or antigen-binding portion thereof, such that animmune response to the antigen in the subject is enhanced. Preferably,the antibody is a human anti-human LAG3 antibody (such as any of thehuman anti-LAG3 antibodies described herein). The antigen can be, forexample, a tumor antigen, a viral antigen, a bacterial antigen or anantigen from a pathogen. Non-limiting examples of such antigens includethose discussed in the sections above, such as the tumor antigens (ortumor vaccines) discussed above, or antigens from the viruses, bacteriaor other pathogens described above.

Combination Therapy

A LAG3 binding polypeptide, e.g., an anti-LAG3 antibody or antibodyfragment, can be administered alone or in combination with one or moreadditional therapies such as chemotherapy radiotherapy, immunotherapy,surgical intervention, or any combination of these. Long-term therapy isequally possible as is adjuvant therapy in the context of othertreatment strategies, as described above.

In certain embodiments of such methods, one or more polypeptidetherapeutic agents can be administered, together (simultaneously) or atdifferent times (sequentially). In addition, polypeptide therapeuticagents can be administered with another type of compounds for treatingcancer or for inhibiting angiogenesis.

The disclosed human anti-LAG-3 antibodies can be co-administered withone or other more therapeutic agents, e.g., a cytotoxic agent, aradiotoxic agent or an immunosuppressive agent. The antibody can belinked to the agent (as an immuno-complex) or can be administeredseparate from the agent. In the latter case (separate administration),the antibody can be administered before, after or concurrently with theagent or can be co-administered with other known therapies, e.g., ananti-cancer therapy, e.g., radiation. Such therapeutic agents include,among others, anti-neoplastic agents such as doxorubicin (adriamycin),cisplatin bleomycin sulfate, carmustine, chlorambucil, dacarbazine andcyclophosphamide hydroxyurea which, by themselves, are only effective atlevels which are toxic or subtoxic to a patient. Cisplatin isintravenously administered as a 100 mg/ml dose once every four weeks andadriamycin is intravenously administered as a 60-75 mg/ml dose onceevery 21 days. Coadministration of the anti-LAG3 antibodies and antibodyfragments of the invention, with chemotherapeutic agents provides twoanti-cancer agents which operate via different mechanisms which yield acytotoxic effect to human tumor cells. Such co-administration can solveproblems due to development of resistance to drugs or a change in theantigenicity of the tumor cells which would render them unreactive withthe antibody.

An anti-LAG3 antibody or antibody fragment as described herein, may becoadministered with one or more additional antibodies that are effectivein stimulating immune responses to thereby further enhance, stimulate orupregulate immune responses in a subject. For example, the inventionprovides a method for stimulating an immune response in a subjectcomprising administering to the subject an anti-LAG3 antibody orantibody fragment and one or more additional immunostimulatoryantibodies, such as an anti-PD-1 antibody, an anti-PD-L1 antibody and/oran anti-CTLA-4 antibody, such that an immune response is stimulated inthe subject, for example to inhibit tumor growth or to stimulate ananti-viral response.

An important part of the immune system is its ability to distinguishbetween normal cells in the body and those it sees as “foreign.” Thislets the immune system attack the foreign cells while leaving the normalcells alone. To do this, it uses “checkpoints,” which are molecules oncertain immune cells that need to be activated (or inactivated) to startan immune response. Cancer cells sometimes find ways to use thesecheckpoints to avoid being attacked by the immune system. Accordingly,an immune checkpoint inhibitor includes a drug or agent, e.g., anantibody, that can activate T cells which are inactive in the absence ofthe drug or agent due, at least in part, to signaling from a cancer cellwhich can maintain the inactive state of the T cell.

Thus, in one embodiment, an anti-LAG3 antibody or antigen bindingantibody fragment of the invention is used in combination with an immunecheckpoint inhibitor for the treatment of cancer. For example, in oneembodiment, an anti-LAG3 antibody, or antigen binding fragment,described herein is administered in combination with an antibody whichis an immune checkpoint inhibitor, including, but not limited to, ananti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) antibody, ananti-programmed death 1 (PD-1) antibody, or an anti-programmeddeath-ligand 1 (PD-L1) antibody. In one embodiment, an anti-LAG3antibody, or antigen binding fragment, described herein is administeredin combination with trastuzumab (Herceptin).

In one embodiment, the subject is administered an anti-LAG3 antibody orantibody fragment and an anti-PD-1 antibody. In another embodiment, thesubject is administered an anti-LAG3 antibody or antibody fragment andan anti-PD-L1 antibody. In yet another embodiment, the subject isadministered an anti-LAG-3 antibody or antibody fragment and ananti-CTLA-4 antibody.

In one embodiment, the invention provides a method for treating ahyperproliferative disease (e.g., cancer), comprising administering aLAG3 antibody and a CTLA-4 antibody to a subject. In furtherembodiments, the anti-LAG3 antibody is administered at a subtherapeuticdose, the anti-CTLA-4 antibody is administered at a subtherapeutic dose,or both are administered at a subtherapeutic dose. Alternatively, amethod for altering an adverse event associated with treatment of ahyperproliferative disease with an immunostimulatory agent, comprisingadministering an anti-LAG3 antibody and a subtherapeutic dose ofanti-CTLA-4 antibody to a subject. In one embodiment, an anti-LAG3antibody, or antigen binding fragment, described herein is administeredin combination with an anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4)antibody, for example ipilimumab (YERVOY) or tremelimumab (CP-675,206;MedImmune).

Another combination comprises administering a LAG3 antibody or antibodyfragment and a PD-1 or PD-L1 antibody to a subject. In one embodiment,an anti-LAG3 antibody, or antigen binding fragment, described herein isadministered in combination with an anti-programmed death 1 (PD-1)antibody, for example pembrolizumab (KEYTRUDA) or nivolumab (OPDIVO). Inone embodiment, an anti-LAG3 antibody, or antigen binding fragment,described herein is administered in combination with an anti-programmeddeath-ligand 1 (PD-L1) antibody, for example avelumab (MSB0010718C),atezolizumab (TECENTRIQ) or durvalumab (MEDI4736). In furtherembodiments, the anti-LAG-3 antibody is administered at a subtherapeuticdose, the anti-PD-1 or PD-L1 antibody is administered at asubtherapeutic dose, or both are administered at a subtherapeutic dose.

Blockade of LAG3 and one or more second target antigens such as CTLA-4and/or PD-1 and/or PD-L1 by antibodies can enhance the immune responseto cancerous cells in the patient. Cancers whose growth may be inhibitedusing the antibodies of the instant disclosure include cancers typicallyresponsive to immunotherapy. Representative examples of cancers fortreatment with the combination therapy of the instant disclosure includethose cancers specifically listed above in the discussion of monotherapywith anti-LAG3 antibodies.

Therapeutic Methods and Compositions

Suitable routes of administering the antibody compositions describedherein (e.g., human monoclonal antibodies, multispecific and bispecificmolecules and immunoconjugates) are in vivo and in vitro are well knownin the art and can be selected by those of ordinary skill. For example,the antibody compositions can be administered by injection (e.g.,intravenous or subcutaneous). Suitable dosages of the molecules usedwill depend on the age and weight of the subject and the concentrationand/or formulation of the antibody composition.

Techniques and dosages for administration vary depending on the type ofspecific polypeptide and the specific condition being treated but can bereadily determined by the skilled artisan. In general, regulatoryagencies require that a protein reagent to be used as a therapeutic isformulated so as to have acceptably low levels of pyrogens. Accordingly,therapeutic formulations will generally be distinguished from otherformulations in that they are substantially pyrogen free, or at leastcontain no more than acceptable levels of pyrogen as determined by theappropriate regulatory agency (e.g., FDA).

Therapeutic compositions of the present disclosure may be administeredwith a pharmaceutically acceptable diluent, carrier, or excipient, inunit dosage form. Administration may be parenteral (e.g., intravenous,subcutaneous), oral, or topical, as non-limiting examples. In addition,any gene therapy technique, using nucleic acids encoding thepolypeptides of the invention, may be employed, such as naked DNAdelivery, recombinant genes and vectors, cell-based delivery, includingex vivo manipulation of patients' cells, and the like.

The composition can be in the form of a pill, tablet, capsule, liquid,or sustained release tablet for oral administration; or a liquid forintravenous, subcutaneous or parenteral administration; gel, lotion,ointment, cream, or a polymer or other sustained release vehicle forlocal administration.

Methods well known in the art for making formulations are found, forexample, in “Remington: The Science and Practice of Pharmacy” (20th ed.,ed. A. R. Gennaro A R., 2000, Lippincott Williams & Wilkins,Philadelphia, Pa.). Formulations for parenteral administration may, forexample, contain excipients, sterile water, saline, polyalkylene glycolssuch as polyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds.Nanoparticulate formulations (e.g., biodegradable nanoparticles, solidlipid nanoparticles, liposomes) may be used to control thebiodistribution of the compounds. Other potentially useful parenteraldelivery systems include ethylene-vinyl acetate copolymer particles,osmotic pumps, implantable infusion systems, and liposomes. Theconcentration of the compound in the formulation varies depending upon anumber of factors, including the dosage of the drug to be administered,and the route of administration.

The polypeptide may be optionally administered as a pharmaceuticallyacceptable salt, such as non-toxic acid addition salts or metalcomplexes that are commonly used in the pharmaceutical industry.Examples of acid addition salts include organic acids such as acetic,lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic,palmitic, suberic, salicylic, tartaric, methanesulfonic,toluenesulfonic, or trifluoroacetic acids or the like; polymeric acidssuch as tannic acid, carboxymethyl cellulose, or the like; and inorganicacid such as hydrochloric acid, hydrobromic acid, sulfuric acidphosphoric acid, or the like. Metal complexes include zinc, iron, andthe like. In one example, the polypeptide is formulated in the presenceof sodium acetate to increase thermal stability.

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose and sorbitol), lubricating agents, glidants, andanti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid,silicas, hydrogenated vegetable oils, or talc).

Formulations for oral use may also be provided as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent, or as soft gelatin capsules wherein the activeingredient is mixed with water or an oil medium.

A therapeutically effective dose refers to a dose that produces thetherapeutic effects for which it is administered. The exact dose willdepend on the disorder to be treated, and may be ascertained by oneskilled in the art using known techniques. In general, the polypeptideis administered at about 0.01 μg/kg to about 50 mg/kg per day,preferably 0.01 mg/kg to about 30 mg/kg per day, most preferably 0.1mg/kg to about 20 mg/kg per day. The polypeptide may be given daily(e.g., once, twice, three times, or four times daily) or preferably lessfrequently (e.g., weekly, every two weeks, every three weeks, monthly,or quarterly). In addition, as is known in the art, adjustments for ageas well as the body weight, general health, sex, diet, time ofadministration, drug interaction, and the severity of the disease may benecessary, and will be ascertainable with routine experimentation bythose skilled in the art.

Preferably, the disclosed antibodies are administered by inhalation, butaerosolization of full IgG antibodies may prove limiting due to theirmolecular size (˜150 kDa). To maximize available commercialaerosolization devices, smaller Fab fragments may be required.

In certain embodiments, the subject anti-LAG3 antibodies or antibodyfragments of the invention can be used alone.

Diagnostics and Kits

In certain embodiments, the binding polypeptides, e.g., antibodies, orfragments thereof can be labeled or unlabeled for diagnostic purposes.Typically, diagnostic assays entail detecting the formation of a complexresulting from the binding of a binding polypeptide, e.g., an antibody,to LAG3. The binding polypeptides or fragments can be directly labeled,similar to antibodies. A variety of labels can be employed, including,but not limited to, radionuclides, fluorescers, enzymes, enzymesubstrates, enzyme cofactors, enzyme inhibitors and ligands (e.g.,biotin, haptens). Numerous appropriate immunoassays are known to theskilled artisan (see, for example, U.S. Pat. Nos. 3,817,827; 3,850,752;3,901,654; and 4,098,876). When unlabeled, the binding polypeptides canbe used in assays, such as agglutination assays. Unlabeled bindingpolypeptides, e.g., antibodies or fragments thereof, can also be used incombination with another (one or more) suitable reagent which can beused to detect the binding polypeptide, such as a labeled antibodyreactive with the binding polypeptide or other suitable reagent (e.g.,labeled protein A).

In one embodiment, the binding polypeptides, e.g., antibodies orfragments thereof, of the present invention can be utilized in enzymeimmunoassays, wherein the subject polypeptides are conjugated to anenzyme. When a biological sample comprising a LAG3 protein is combinedwith the subject binding polypeptides, binding occurs between thebinding polypeptides and the LAG3 protein. In one embodiment, a samplecontaining cells expressing a LAG3 protein (e.g., endothelial cells) iscombined with the subject antibodies, and binding occurs between thebinding polypeptides and cells bearing a LAG3 protein recognized by thebinding polypeptide. These bound cells can be separated from unboundreagents and the presence of the binding polypeptide-enzyme conjugatespecifically bound to the cells can be determined, for example, bycontacting the sample with a substrate of the enzyme which produces acolor or other detectable change when acted on by the enzyme. In anotherembodiment, the subject binding polypeptides can be unlabeled, and asecond, labeled polypeptide (e.g., an antibody) can be added whichrecognizes the subject binding polypeptide.

In certain aspects, kits for use in detecting the presence of a LAG3protein in a biological sample using the antibodies or fragments thereofof the invention can also be prepared. Such kits will include a LAG3binding polypeptide, e.g., antibodies or fragments thereof, which bindsto a LAG3 protein or portion of said receptor, as well as one or moreancillary reagents suitable for detecting the presence of a complexbetween the binding polypeptide and the receptor protein or portionsthereof. The polypeptide compositions of the present invention can beprovided in lyophilized form, either alone or in combination withadditional antibodies specific for other epitopes. The bindingpolypeptides and/or antibodies, which can be labeled or unlabeled, canbe included in the kits with adjunct ingredients (e.g., buffers, such asTris, phosphate and carbonate, stabilizers, excipients, biocides and/orinert proteins, e.g., bovine serum albumin) For example, the bindingpolypeptides and/or antibodies can be provided as a lyophilized mixturewith the adjunct ingredients, or the adjunct ingredients can beseparately provided for combination by the user. Generally these adjunctmaterials will be present in less than about 5% weight based on theamount of active binding polypeptide or antibody, and usually will bepresent in a total amount of at least about 0.001% weight based onpolypeptide or antibody concentration. Where a second antibody capableof binding to the binding polypeptide is employed, such antibody can beprovided in the kit, for instance in a separate vial or container. Thesecond antibody, if present, is typically labeled, and can be formulatedin an analogous manner with the antibody formulations described above.

The invention is further described in the following examples, which arein not intended to limit the scope of the invention.

Example 1

A screen was performed to identify human anti-human LAG3 antibodies, theheavy and light chain variable amino acid sequences (including the CDRsthereof).

To determine the binding capability of various anti-LAG3 antibodiesdisclosed herein, T cells were cultured with magnetic beads coated withantibodies reactive with CD3 and CD28. After three days of culture asignificant percentage of the cells expressed LAG3. The LAG3 expressingcells were incubated with the test antibodies (1 microgram per ml)followed by staining with a phycoerythrin labeled goat anti-human IgGantibody. Several antibodies had reactivity with the activated T cellsand these are shown in FIG. 1.

An analysis of the cross-reactivity of various LAG3 antibodies torecombinant mouse LAG3 and human LAG3 was also performed. A MaxisorbELISA plate was coated with 2 ug/mL recombinant human, and mouse LAG3/Fc(blank: PBS). Incubated overnight at 4° C. The plate was washed 3 timeswith PBS-Tween (PBST), then blocked with Casein blocking buffer for 1hour at room temperature. Next, IgGs diluted in casein (about 5 ug/ml)were added, and incubated 30 min with shaking. The plate was washed 3times with PBST. Horseradish peroxidase (HRP)-conjugated goat anti-humanLambda HRP (1:1000 in casein) was added, then3,3′,5,5′-Tetramethylbenzidine (TMB) was added as substrate anddeveloped 30 min 2M H2SO4 was used to stop the reaction and the OD wasread at 450 nm Anti-AIP antibody C7 was used as a control antibody. Theresults are provided in FIG. 2 and show that anti-LAG3 antibodies L35G6,L33H11, L35D4, L32A9, L32A4 and L32D10 bind to human but do not bind tomouse LAG3.

The binding affinity of antibody L3C5 for human LAG3 was also testedusing a BiaCore assay. For antibody L3C5, K_(a) was found to be 4.73 E5(1/Ms), K_(d) was found to be 0.0717 (1/s), R^(max) was found to be261(RU), K_(A) was found to be 6.6 E6 (1/M), K_(D) was found to be 1.52E-7 (M) and chi2 was 3.65. Biacore was used to measure the affinity ofLAG3 antibody L3C5. Anti-human Fc antibody (GE, BR-1008-39) wasimmobilized on CM5 sensor chip to approximately 5000 RU using standardNHS/EDC coupling methodology. Antibody (approximately 2 ug/ml) werecaptured for 60 s at a flow rate 10 uL/min. Recombinant human LAG3/Hiswas serially diluted in running buffer (HBS-EP). All measurements wereconducted with a flow rate of 30 μL/min Surfaces were regenerated with3M MgCl2 (from human antibody kit) for 60 s. A 1:1 (Langmuir) bindingmodel was used to fit the data.

Example 2

Functional in vitro studies using a two-step activation protocol mixedlymphocyte reactions (MLR) to measure T cell activation were performed.The functional activity of the anti-LAG3 antibodies was evaluated bymeasuring their effect on the response of LAG3 expressing T cells tostimulation by the superantigen, staphylococcal enterotoxin B (SEB). Tcells were cultured with magnetic beads coated with antibodies reactivewith CD3 and CD28. The following day, the beads were removed and thecells were cultured in fresh medium. After a further two days the Tcells were harvested and added to the wells of a flat bottom microtiterplate at a concentration of 1×10⁵ cells per well. To these wells wereadded 2×10⁴ freshly prepared B cells and SEB (10 ng/ml). After threedays of culture the cells were stained for CD25 expression. To determinethe effect of the anti-LAG3 antibodies, the percent change with respectto the medium control was calculated and is shown in FIG. 3. FIG. 4shows the level of T cell activation, as measured by CD25 expression, inthe presence of the anti-LAG3 antibodies. This data is expressed as apercent change from that of medium control and is shown in FIG. 3. FIGS.3 and 4 together show that four out of the five anti-LAG3 antibodiestested augment T cell activation greater than that that of control IgG.Thus, the antibodies were able to block LAG3 activity and promote T cellactivation.

Example 3

Functional in vitro studies using a mixed lymphocyte reactions (MLR)were performed to evaluate other anti-LAG3 antibodies, with cytokineproduction being the measure of T cell activation. An ELISA assay wascarried out to determine the effect of anti-LAG3 antibodies L32D10,L3E3, L3C5 and L3A1 (at concentrations of 5 μg/ml and 0.5 μg/ml) on IL-2and interferon gamma (IFNγ) cytokine production. IL-2 and IFNγ cytokineproduction are measures of T-cell activation. ELISA kits were purchasedfrom Biolegend and were performed following the manufacturer'sinstructions. The results presented in FIG. 5 and FIG. 6 show thatL32D10 and L3E3 augment the production of both IL-2 and interferon gamma(IFNγ), respectively, whereas clone L3A1 only augments IL-2 production.An IgG1 that does not bind to LAG3 and media only were used as controls.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of the appended claims. Allpublications, patents, and patent applications cited herein are herebyincorporated by reference in their entirety for all purposes.

TABLE 3 Sequence Listing VH Heavy chain binding VL light chain bindingBinder region region L35D4 QVQLVQSGAEVKKPGASVKVSCKASGYQSVLTQPPSASGSPGQSVTISCTGTS TFTSYYMHWVRQAPGQGLEWMGIINPSSDVGGYNYVSWYQQYPGKAPRLMIFE AGSTSYAQKFQGRVTMTRDTSTSTVYMVTERASGVPDRFSGSKSGNTASLTVS ELSSLRSEDTAVYYCARELMATGGFDYGLQTEDEAVYFCSSYSGSNNPGAMFG WGQGTLVTVSS SEQ ID NO. 1GGTKLTVL SEQ ID NO. 2 L35D4 HC CDR1: SYYMH LC CDR1: TGTSSDVGGYNYVSSEQ ID NO. 15 SEQ ID NO. 18 HC CDR2: IINPSAGSTSYAQKFQG LC CDR2: EVTERASSEQ ID NO. 16 SEQ ID NO. 19 HC CDR3: ELMATGGFDY LC CDR3: SSYSGSNNPGAMSEQ ID NO. 17 SEQ ID NO. 20 L35G6 QVQLVQSGAEVKKPGASVKVSCKASGYQAGLTQPASVSGSPGQSITISCTGSS TFTSYYMHWVRQAPGQGLEWMGIINPSSDVGGYSYVSWYQKHPGKAPKLMIYD AGSTSYAQKFQGRVTMTRDTSTSTVYMVTNRPSGVSNRFSGSKSGNTASLTIS ELSSLRSEDTAVYYCARELMATGGFDYGLQAEDEADYYCSTYTRSNTLVFGPG WGQGTLVTVSS SEQ ID NO. 1 TKVTVL SEQ ID NO. 3L35G6 HC CDR1: SYYMH HC CDR1: TGSSSDVGGYSYVS SEQ ID NO. 15 SEQ ID NO. 21HC CDR2: IINPSAGSTSYAQKFQG HC CDR2: DVTNRPS SEQ ID NO. 16 SEQ ID NO. 22HC CDR3: ELMATGGFDY HC CDR3: STYTRSNTLV SEQ ID NO. 17 SEQ ID NO. 23L33H11 QVQLVQSGAEVKKPGASVKVSCKASGY LPVLTQPASVSGSPGQSITISCTGTSTFTSYYMHWVRQAPGQGLEWMGIINPS SDVGGYNYVSWYQQHPGKAPKLMIYDAGSTSYAQKFQGRVTMTRDTSTSTVYM VTNRPSGVSNRFSGSKSGNTASLTISELSSLRSEDTAVYYCARELMATGGFDY GLQAEDEADYYCSSYTSSNTLLFGGGWGQGTLVTVSS SEQ ID NO. 1 TQLTVL SEQ ID NO. 4 L33H11 HC CDR1: SYYMHHC CDR1: TGTSSDVGGYNYVS SEQ ID NO. 15 SEQ ID NO. 24HC CDR2: IINPSAGSTSYAQKFQG HC CDR2: DVTNRPS SEQ ID NO. 16 SEQ ID NO. 25HC CDR3: ELMATGGFDY HC CDR3: SSYTSSNTLL SEQ ID NO. 17 SEQ ID NO. 26L32A9 QVQLVQSGAEVKKPGASVKVSCKASGY QSVVTQPPSVSAAPGQKVTISCSGSSTFTSYYMHWVRQAPGQGLEWMGIINPS SNIGNNYVSWYQQLPGTAPKLLIYDNAGSTSYAQKFQGRVTMTRDTSTSTVYM NKRHSGIPDRFSGSTSDTSATLGITRELSSLRSEDTAVYYCARELMATGGFDY LQTGDEADYYCGTWDSSLSAYVFGTGWGQGTLVTVSS SEQ ID NO. 1 TKVTVL SEQ ID NO. 5 L32A9 HC CDR1: SYYMHHC CDR1: SGSSSNIGNNYVS SEQ ID NO. 15 SEQ ID NO. 27HC CDR2: IINPSAGSTSYAQKFQG HC CDR2: DNNKRHS SEQ ID NO. 16 SEQ ID NO. 28HC CDR3: ELMATGGFDY HC CDR3: GTWDSSLSAYV SEQ ID NO. 17 SEQ ID NO. 29L32D10 QVQLVQSGAEVKKPGASVKVSCKASGY QSVLTQPPSASGSPGQSVTISCTGTSTFTSYYMHWVRQAPGQGLEWMGIINPS SDVGGYDYVSWYQQHQGKAPKLMIYDAGSTSYAQKFQGRVTMTRDTSTSTVYM VSNRPSGVSNRFSGSKSGNTASLTISELSSLRSEDTAVYYCARELMATGGFDY GLQAEDEADYYCSSYTSSTTLVFGGGWGQGTLVTVSS SEQ ID NO. 1 TKLTVL SEQ ID NO. 6 L32D10 HC CDR1: SYYMHHC CDR1: TGTSSDVGGYDYVS SEQ ID NO. 15 SEQ ID NO. 30HC CDR2: IINPSAGSTSYAQKFQG HC CDR2: DVSNRPS SEQ ID NO. 16 SEQ ID NO. 31HC CDR3: ELMATGGFDY HC CDR3: SSYTSSTTLV SEQ ID NO. 17 SEQ ID NO. 32L32A4 QVQLVQSGAEVKKPGASVKVSCKASGY QSVLTQPASVSGSPGQSITISCTGTSTFTSYYMHWVRQAPGQGLEWMGIINPS SDIGAYNFVSWYQQHPGKAPKLMIYGAGSTSYAQKFQGRVTMTRDTSTSTVYM VSNRPSGVSSRFSGSKSGSTASLTISELSSLRSEDTAVYYCARELMATGGFDY GLQAEDEADYYCSSYTTSGSAVFGTGWGQGTLVTVSS SEQ ID NO. 1 TKLTVL SEQ ID NO. 7 L32A4 HC CDR1: SYYMHHC CDR1: TGTSSDIGAYNFVS SEQ ID NO. 15 SEQ ID NO. 33HC CDR2: IINPSAGSTSYAQKFQG HC CDR2: GVSNRPS SEQ ID NO. 16 SEQ ID NO. 34HC CDR3: ELMATGGFDY HC CDR3: SSYTTSGSAV SEQ ID NO. 17 SEQ ID NO. 35 L3A1EVQLLESGAEVKKPGASVKVSCKASGY QSVLTQPASVSGSPGQSITISCTGTSTFTSYYMHWVRQAPGQGLEWMGIINPS SDIGAYNFVSWYQQHPGKAPKLMIYGAGSTSYAQKFQGRVTMTRDTSTSTVYM VSNRPSGVSSRFSGSKSGSTASLTITELSSLRSEDTAVYYCARELMATGGFDY GLQAEDEADYYCSSYTTSGSAVFGTGWGQGTLVTVSS SEQ ID NO. 8 TKLTVL SEQ ID NO. 9 L3A1 HC CDR1: SYYMHHC CDR1: TGTSSDIGAYNFVS SEQ ID NO. 36 SEQ ID NO. 39HC CDR2: IINPSAGSTSYAQKFQG HC CDR2: GVSNRPS SEQ ID NO. 37 SEQ ID NO. 40HC CDR3: ELMATGGFDY HC CDR3: SSYTTSGSAV SEQ ID NO. 38 SEQ ID NO. 41L3A10 EVQLLESGGGVVQPGRSLRVSCAASGF DVVMTQSPSSLSASVGDRVSITCRASTFSNHAMHWVRQAPGKGLEWVAVISYD QNIGRYLNWYQQKPGKAPKLLVSAASGSKKFYSDSVRGRFTISRDNSKNTLYL SLQGGVPSRFSGSGSGTDFTLTISRLQMNSLRPEDTAVYYCAKGAHGYTSGWH QPEDFATYFCQQTYSSPQCTFGQGTK DYWGQGTLVTVSSVDIK SEQ ID NO. 11 SEQ ID NO. 10 L3A10 HC CDR1: NHAMHHC CDR1: RASQNIGRYLN SEQ ID NO. 42 SEQ ID NO. 45HC CDR2: VISYDGSKKFYSDSVRG HC CDR2: AASSLQG SEQ ID NO. 43 SEQ ID NO. 46HC CDR3: GAHGYTSGWHDY HC CDR3: QQTYSSPQCT SEQ ID NO. 44 SEQ ID NO. 47L3C5 QVQLVQSGSELKKPGASVKVSCKASGY QSVLTQPASVSGSPGQSITISCTGTSTFTNYYMHWVRQAPGQGLEWMGIINPS SDVGGYNYVSWYQQHPGKAPKLMIYDGGATNYAQKFQGRVTMTRDTSTSTVYM VSNRPSGASNRFSGSKSGNTASLTISELSSLRSEDTAVYYCARDSGYDLGYGM GLQAEDEADYYCSSYTNRNTLLFGGG DVWGQGTLVTVSSTKLTVL SEQ ID NO. 13 SEQ ID NO. 12 L3C5 HC CDR1: NYYMHHC CDR1: TGTSSDVGGYNYVS SEQ ID NO. 48 SEQ ID NO. 51HC CDR2: IINPSGGATNYAQKFQG HC CDR2: DVSNRPS SEQ ID NO. 49 SEQ ID NO. 52HC CDR3: DSGYDLGYGMDV HC CDR3: SSYTNRNTLL SEQ ID NO. 50 SEQ ID NO. 53L3E3 EVQLLESGAEVKKPGASVKVSCKASGY QSVLTQPASASGSPGQSITISCTGTSTFTSYYMHWVRQAPGQGLEWMGIINPS SDVGGYNYVSWYQQHPGKAPKLMIYDAGSTSYAQKFQGRVTMTRDTSTSTVYM VSNRPSGVSNRFSGSKSGNTASLTISELSSLRSEDTAVYYCARELMATGGFDY GLQAEDEANYYCSSYTSSSTNVFGTGWGQGTLVTVSS SEQ ID NO. 8 TKVTVL SEQ ID NO. 14 L3E3 HC CDR1: SYYMHHC CDR1: TGTSSDVGGYNYVS SEQ ID NO. 36 SEQ ID NO. 54HC CDR2: IINPSAGSTSYAQKFQG HC CDR2: DVSNRPS SEQ ID NO. 37 SEQ ID NO. 55HC CDR3: ELMATGGFDY HC CDR3: SSYTSSSTNV SEQ ID NO. 38 SEQ ID NO. 56

We claim:
 1. An isolated anti-human LAG3 (hLAG3) antibody, or anantigen-binding fragment thereof, comprising a heavy chain variabledomain comprising a heavy chain CDR set (CDR1, CDR2, and CDR3) as setforth in SEQ ID Nos: 36, 37, and 38; and a light chain variable domaincomprising a light chain CDR set (CDR1, CDR2, and CDR3) as set forth inSEQ ID Nos: 54, 55, and
 56. 2. The anti-hLAG3 antibody, or theantigen-binding fragment thereof, of claim 1, wherein the heavy chainvariable domain comprises the amino acid sequence that is at least 95%identical to SEQ ID NO. 8, and wherein the light chain variable domaincomprises the amino acid sequence that is at least 95% identical to SEQID NO.
 14. 3. The anti-hLAG3 antibody, or the antigen-binding fragmentthereof, of claim 1, wherein the heavy chain variable domain comprisesthe amino acid sequence set forth in SEQ ID NO. 8; and wherein the lightchain variable domain comprises the amino acid sequence set forth in SEQID NO.
 14. 4. The anti-hLAG antibody, or the antigen binding fragmentthereof, of claim 1, wherein the antibody has a K_(D) of at least 1×10⁻⁶M.
 5. The anti-hLAG3 antibody, or the antigen-binding fragment thereof,of claim 1, wherein the antibody is an IgG, an IgM, an IgD, an IgA, oran IgE.
 6. The anti-hLAG3 antibody, or the antigen-binding fragmentthereof, of claim 5, wherein the antibody is an IgG1 or an IgG4 isotype.7. A pharmaceutical composition comprising the anti-hLAG3 antibody, orthe antigen binding fragment thereof of claim 1, and a pharmaceuticallyacceptable carrier.
 8. The anti-hLAG3 antibody, or the antigen-bindingfragment thereof, of claim 1, wherein the antibody, or theantigen-binding fragment thereof, is a monoclonal antibody, a humanantibody, a humanized antibody, an Fab, an Fab′, an F(ab′)2, an Fv, adomain antibody (dAb), a single-chain antibody (scFv), a chimericantibody, a diabody, a triabody or a tetrabody.
 9. The anti-hLAG3antibody, or the antigen-binding fragment thereof, of claim 2, whereinthe antibody, or the antigen-binding fragment thereof, is a monoclonalantibody, a human antibody, a humanized antibody, an Fab, an Fab′, anF(ab′)2, an Fv, a domain dAb, an scFv, a chimeric antibody, a diabody, atriabody or a tetrabody.
 10. The anti-hLAG3 antibody, or theantigen-binding fragment thereof, of claim 3, wherein the antibody, orthe antigen-binding fragment thereof, is a monoclonal antibody, a humanantibody, a humanized antibody, an Fab, an Fab′, an F(ab′)2, an Fv, adomain dAb, an scFv, a chimeric antibody, a diabody, a triabody or atetrabody.